444 lines
16 KiB
Python
444 lines
16 KiB
Python
import logging
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import time
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from contextlib import contextmanager
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from typing import Callable, List, Optional, Tuple
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import numpy as np
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from labthings import current_action, fields, find_component
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from labthings.extensions import BaseExtension
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from labthings.views import ActionView, View
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from scipy import ndimage
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from openflexure_microscope.camera.base import BaseCamera
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from openflexure_microscope.devel import abort
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from openflexure_microscope.microscope import Microscope
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from openflexure_microscope.stage.base import BaseStage
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from openflexure_microscope.utilities import set_properties
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### Autofocus utilities
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class JPEGSharpnessMonitor:
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def __init__(self, microscope: Microscope):
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self.microscope: Microscope = microscope
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self.camera: BaseCamera = microscope.camera
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self.stage: BaseStage = microscope.stage
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self.recording_start_time: Optional[float] = None
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self.stage_positions: List[Tuple[int, int, int]] = []
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self.stage_times: List[float] = []
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self.jpeg_times: List[float] = []
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self.jpeg_sizes: List[int] = []
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def start(self):
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# Log the recording start time
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self.recording_start_time = time.time()
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def stop(self):
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self.camera.stream.stop_tracking()
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self.camera.stream.reset_tracking()
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def focus_rel(self, dz: int, backlash: bool = False, **kwargs):
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# Store the start time and position
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self.camera.stream.start_tracking()
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self.stage_times.append(time.time())
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self.stage_positions.append(self.stage.position)
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# Main move
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self.stage.move_rel((0, 0, dz), backlash=backlash, **kwargs)
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# Store the end time and position
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self.camera.stream.stop_tracking()
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self.stage_times.append(time.time())
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self.stage_positions.append(self.stage.position)
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# Retrieve frame data
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for frame in self.camera.stream.frames:
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# Make timestamp absolute Unix time
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self.jpeg_times.append(frame.time)
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self.jpeg_sizes.append(frame.size)
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# Clear frame data for this move from the stream
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self.camera.stream.reset_tracking()
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# Index of the data for this movement
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data_index = len(self.stage_positions) - 2
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# Final z position after move
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final_z_position = self.stage_positions[-1][2]
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return data_index, final_z_position
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def move_data(self, istart: int, istop: Optional[int] = None):
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"""Extract sharpness as a function of (interpolated) z"""
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if istop is None:
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istop = istart + 2
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jpeg_times = np.array(self.jpeg_times)
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jpeg_sizes = np.array(self.jpeg_sizes)
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stage_times = np.array(self.stage_times)[istart:istop]
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stage_zs = np.array(self.stage_positions)[istart:istop, 2]
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try:
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start = np.argmax(jpeg_times > stage_times[0])
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stop = np.argmax(jpeg_times > stage_times[1])
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except ValueError as e:
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if np.sum(jpeg_times > stage_times[0]) == 0:
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raise ValueError(
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"No images were captured during the move of the stage. Perhaps the camera is not streaming images?"
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) from e
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else:
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raise e
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if stop < 1:
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stop = len(jpeg_times)
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logging.debug("changing stop to %s", (stop))
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jpeg_times = jpeg_times[start:stop]
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jpeg_zs = np.interp(jpeg_times, stage_times, stage_zs)
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return jpeg_times, jpeg_zs, jpeg_sizes[start:stop]
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def sharpest_z_on_move(self, index: int):
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"""Return the z position of the sharpest image on a given move"""
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_, jz, js = self.move_data(index)
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if len(js) == 0:
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raise ValueError(
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"No images were captured during the move of the stage. Perhaps the camera is not streaming images?"
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)
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return jz[np.argmax(js)]
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def data_dict(self):
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"""Return the gathered data as a single convenient dictionary"""
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data = {}
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for k in ["jpeg_times", "jpeg_sizes", "stage_times", "stage_positions"]:
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data[k] = getattr(self, k)
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return data
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@contextmanager
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def monitor_sharpness(microscope: Microscope):
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m = JPEGSharpnessMonitor(microscope)
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m.start()
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try:
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yield m
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finally:
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m.stop()
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def sharpness_sum_lap2(rgb_image: np.ndarray):
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"""Return an image sharpness metric: sum(laplacian(image)**")"""
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image_bw = np.mean(rgb_image, 2)
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image_lap = ndimage.filters.laplace(image_bw)
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return np.mean(image_lap.astype(np.float) ** 4)
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def sharpness_edge(image: np.ndarray):
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"""Return a sharpness metric optimised for vertical lines"""
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gray = np.mean(image.astype(float), 2)
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n = 20
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edge = np.array([[-1] * n + [1] * n])
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return np.sum(
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[np.sum(ndimage.filters.convolve(gray, W) ** 2) for W in [edge, edge.T]]
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)
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### Autofocus extension
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class AutofocusExtension(BaseExtension):
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def __init__(self):
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super().__init__(
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"org.openflexure.autofocus",
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version="2.0.0",
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description="Actions to move the microscope in Z and pick the point with the sharpest image.",
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)
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self.add_view(
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MeasureSharpnessAPI, "/measure_sharpness", endpoint="measure_sharpness"
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)
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self.add_view(AutofocusAPI, "/autofocus", endpoint="autofocus")
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self.add_view(FastAutofocusAPI, "/fast_autofocus", endpoint="fast_autofocus")
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self.add_view(
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UpDownUpAutofocusAPI, "/updownup_autofocus", endpoint="updownup_autofocus"
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)
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self.add_view(
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MoveAndMeasureAPI, "/move_and_measure", endpoint="move_and_measure"
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)
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def measure_sharpness(
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self, microscope: Microscope, metric_fn: Callable = sharpness_sum_lap2
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):
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"""Measure the sharpness of the camera's current view."""
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if hasattr(microscope.camera, "array") and callable(
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getattr(microscope.camera, "array")
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):
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return metric_fn(getattr(microscope.camera, "array")(use_video_port=True))
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else:
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raise RuntimeError(f"Object {microscope.camera} has no method `array`")
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def autofocus(
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self,
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microscope: Microscope,
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dz: List[int],
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settle: float = 0.5,
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metric_fn: Callable = sharpness_sum_lap2,
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):
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"""Perform a simple autofocus routine.
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The stage is moved to z positions (relative to current position) in dz,
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and at each position an image is captured and the sharpness function
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evaulated. We then move back to the position where the sharpness was
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highest. No interpolation is performed.
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dz is assumed to be in ascending order (starting at -ve values)
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"""
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camera = microscope.camera
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stage = microscope.stage
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with set_properties(stage, backlash=256), stage.lock, camera.lock:
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sharpnesses = []
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positions = []
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# Some cameras may not have annotate_text. Reset if it does
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if getattr(camera, "annotate_text"):
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setattr(camera, "annotate_text", "")
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for _ in stage.scan_z(dz, return_to_start=False):
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if current_action() and current_action().stopped:
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return
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positions.append(stage.position[2])
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time.sleep(settle)
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sharpnesses.append(self.measure_sharpness(microscope, metric_fn))
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newposition = positions[np.argmax(sharpnesses)]
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stage.move_rel((0, 0, newposition - stage.position[2]))
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return positions, sharpnesses
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def move_and_find_focus(self, microscope: Microscope, dz: int):
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"""Make a relative Z move and return the peak sharpness position"""
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with monitor_sharpness(microscope) as m:
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m.focus_rel(dz)
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return m.sharpest_z_on_move(0)
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def move_and_measure(self, microscope: Microscope, dz: int):
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"""Make a relative Z move and return the sharpness data"""
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with monitor_sharpness(microscope) as m:
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m.focus_rel(dz)
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return m.data_dict()
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def fast_autofocus(self, microscope: Microscope, dz: int = 2000):
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"""Perform a down-up-down-up autofocus"""
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with microscope.camera.lock, microscope.stage.lock:
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with monitor_sharpness(microscope) as m:
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# Move to (-dz / 2)
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m.focus_rel(-dz / 2)
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# Move to dz while monitoring sharpness
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# i: Sharpness monitor index for this move
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# z: Final z position after move
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i, z = m.focus_rel(dz)
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# Get the z position with highest sharpness from the previous move (index i)
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fz = m.sharpest_z_on_move(i)
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# Move all the way to the start so it's consistent
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# Store final absolute z position from this return move
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i, z = m.focus_rel(-dz)
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# Move to the target position fz
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# Can't do absolute move here yet so move by (fz - z)
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m.focus_rel(fz - z)
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# Return all focus data
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return m.data_dict()
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def fast_up_down_up_autofocus(
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self,
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microscope: Microscope,
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dz: int = 2000,
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target_z: int = 0,
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initial_move_up: bool = True,
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mini_backlash: int = 25,
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):
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"""Autofocus by measuring on the way down, and moving back up with feedback.
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This autofocus method is very efficient, as it only passes the peak once.
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The sequence of moves it performs is:
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1. Move to the top of the range `dz/2` (can be disabled)
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2. Move down by `dz` while monitoring JPEG size to find the focus.
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3. Move back up to the `target_z` position, relative to the sharpest image.
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4. Measure the sharpness, and compare against the curve recorded in (2) to \\
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estimate how much further we need to go. Make this move, to reach our \\
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target position.
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Moving back to the target position in two steps allows us to correct for
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backlash, by using the sharpness-vs-z curve as a rough encoder for Z.
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Parameters:
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dz: number of steps over which to scan (optional, default 2000)
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target_z: we aim to finish at this position, relative to focus. This may
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be useful if, for example, you want to acquire a stack of images in Z.
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It is optional, and the default value of 0 will finish at the focus.
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initial_move_up: (optional, default True) set this to `False` to move down
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from the starting position. Mostly useful if you're able to combine
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the initial move with something else, e.g. moving to the next scan point.
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mini_backlash: (optional, default 25) is a small extra move made in step
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3 to help counteract backlash. It should be small enough that you
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would always expect there to be greater backlash than this. Too small
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might slightly hurt accuracy, but is unlikely to be a big issue. Too big
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may cause you to overshoot, which is a problem.
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"""
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with microscope.camera.lock, microscope.stage.lock:
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with monitor_sharpness(microscope) as m:
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# Ensure the MJPEG stream has started
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microscope.camera.start_stream()
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logging.debug("Initial move")
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if initial_move_up:
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m.focus_rel(dz / 2)
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# move down
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logging.debug("Move down")
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i, z = m.focus_rel(-dz)
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# now inspect where the sharpest point is, and estimate the sharpness
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# (JPEG size) that we should find at the start of the Z stack
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_, jz, js = m.move_data(i)
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best_z = jz[np.argmax(js)]
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# now move to the start of the z stack
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logging.debug("Move to the start of the z stack")
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i, z = m.focus_rel(
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best_z + target_z - z + mini_backlash
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) # takes us to the start of the stack
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# We've deliberately undershot - figure out how much further we should move based on the curve
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logging.debug("Calculate remining movement")
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current_js = m.camera.stream.last.size
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imax = np.argmax(js) # we want to crop out just the bit below the peak
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js = js[imax:] # NB z is in DECREASING order
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jz = jz[imax:]
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inow = np.argmax(
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js < current_js
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) # use the curve we recorded to estimate our position
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# So, the Z position corresponding to our current sharpness value is zs[inow]
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# That means we should move forwards, by best_z - zs[inow]
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logging.debug("Correction move")
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correction_move = best_z + target_z - jz[inow]
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logging.debug(
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"Fast autofocus scan: correcting backlash by moving %s steps",
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(correction_move),
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)
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m.focus_rel(correction_move)
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return m.data_dict()
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class MeasureSharpnessAPI(View):
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def post(self):
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microscope = find_component("org.openflexure.microscope")
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if not microscope:
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abort(503, "No microscope connected. Unable to measure sharpness.")
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return {"sharpness": self.extension.measure_sharpness(microscope)}
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class MoveAndMeasureAPI(ActionView):
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"""
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Move the stage and measure dynamic sharpness
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"""
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args = {"dz": fields.Int(required=True)}
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def post(self, args):
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microscope = find_component("org.openflexure.microscope")
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if not microscope:
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abort(503, "No microscope connected. Unable to autofocus.")
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if microscope.has_real_stage():
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# Acquire microscope lock with 1s timeout
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with microscope.camera.lock, microscope.stage.lock:
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return self.extension.move_and_measure(microscope, dz=args.get("dz"))
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else:
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abort(503, "No stage connected. Unable to autofocus.")
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class AutofocusAPI(ActionView):
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"""
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Run a standard autofocus
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"""
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args = {"dz": fields.List(fields.Int())}
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def post(self, args):
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microscope = find_component("org.openflexure.microscope")
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if not microscope:
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abort(503, "No microscope connected. Unable to autofocus.")
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# Figure out the range of z values to use
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dz = np.array(args.get("dz", np.linspace(-300, 300, 7)))
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if microscope.has_real_stage():
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logging.debug("Running autofocus...")
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# return a handle on the autofocus task
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return self.extension.autofocus(microscope, dz)
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else:
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abort(503, "No stage connected. Unable to autofocus.")
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class FastAutofocusAPI(ActionView):
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"""
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Run a fast autofocus
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"""
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args = {"dz": fields.Int(missing=2000, example=2000)}
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def post(self, args):
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microscope = find_component("org.openflexure.microscope")
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if not microscope:
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abort(503, "No microscope connected. Unable to autofocus.")
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if microscope.has_real_stage():
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logging.debug("Running autofocus...")
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# Acquire microscope lock with 1s timeout
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with microscope.lock(timeout=1):
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# Run fast_autofocus
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return self.extension.fast_autofocus(microscope, dz=args.get("dz"))
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else:
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abort(503, "No stage connected. Unable to autofocus.")
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class UpDownUpAutofocusAPI(ActionView):
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"""
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Run a fast up-down-up autofocus
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"""
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args = {
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"dz": fields.Int(missing=2000, example=2000),
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"backlash": fields.Int(missing=25, minimum=0),
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}
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def post(self, args):
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microscope = find_component("org.openflexure.microscope")
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if not microscope:
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abort(503, "No microscope connected. Unable to autofocus.")
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# Figure out the parameters to use
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dz = args.get("dz")
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backlash = args.get("backlash")
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if microscope.has_real_stage():
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logging.debug("Running autofocus...")
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# Acquire microscope lock with 1s timeout
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with microscope.lock(timeout=1):
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# Run fast_up_down_up_autofocus
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return self.extension.fast_up_down_up_autofocus(
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microscope, dz=dz, mini_backlash=backlash
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)
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else:
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abort(503, "No stage connected. Unable to autofocus.")
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