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Added fast autofocus integrated with Z stacking in the scan code

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This commit is contained in:
Richard Bowman 2019-04-08 16:12:31 +01:00
parent 1cf1e40c54
commit 7e83d724b8
2 changed files with 71 additions and 19 deletions
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2
openflexure_microscope/plugins/default/scan/api.py
View file

@ -19,6 +19,7 @@ class TileScanAPI(MicroscopeViewPlugin):
style = payload.param('style', default='raster', convert=str) style = payload.param('style', default='raster', convert=str)
autofocus_dz = payload.param('autofocus_dz', default=50, convert=int) autofocus_dz = payload.param('autofocus_dz', default=50, convert=int)
fast_autofocus = payload.param('fast_autofocus', default=False, convert=bool)
use_video_port = payload.param('use_video_port', default=True, convert=bool) use_video_port = payload.param('use_video_port', default=True, convert=bool)
resize = payload.param('size', default=None) resize = payload.param('size', default=None)
@ -43,6 +44,7 @@ class TileScanAPI(MicroscopeViewPlugin):
use_video_port=use_video_port, use_video_port=use_video_port,
resize=resize, resize=resize,
bayer=bayer, bayer=bayer,
fast_autofocus=fast_autofocus,
metadata=metadata, metadata=metadata,
tags=tags tags=tags
) )

88
openflexure_microscope/plugins/default/scan/plugin.py
View file

@ -101,6 +101,7 @@ class ScanPlugin(MicroscopePlugin):
use_video_port: bool = False, use_video_port: bool = False,
resize: Tuple[int, int] = None, resize: Tuple[int, int] = None,
bayer: bool = False, bayer: bool = False,
fast_autofocus = False,
metadata: dict = {}, metadata: dict = {},
tags: list = []): tags: list = []):
@ -124,6 +125,12 @@ class ScanPlugin(MicroscopePlugin):
else: else:
autofocus_enabled = False autofocus_enabled = False
if fast_autofocus and not hasattr(self.microscope.plugin, 'default_fast_autofocus'):
logging.error("Can't use fast autofocus in the scan - the plugin is missing or disabled.")
fast_autofocus = False
z_stack_dz = grid[2] * step_size[2] if grid[2] > 1 else 0 # shorthand for Z stack range
sweep_to_scan_offset = 50 #TODO: make this a parameter, or calibrate it better! too small isn't a big problem, too big causes issues.
# Construct an x-y grid (worry about z later) # Construct an x-y grid (worry about z later)
x_y_grid = construct_grid( x_y_grid = construct_grid(
initial_position, initial_position,
@ -132,27 +139,62 @@ class ScanPlugin(MicroscopePlugin):
style=style style=style
) )
# Keep the initial Z position the same as our current position
next_z = initial_position[2]
if fast_autofocus: # If fast autofocus is enabled, make
next_z += autofocus_dz/2 # sure we start from the top of the range
initial_z = next_z # Save this value for use in raster scans
# Now step through each point in the x-y coordinate array # Now step through each point in the x-y coordinate array
for line in x_y_grid: for line in x_y_grid:
# If rastering, rather than snake (or eventually spiral) # If rastering, rather than snake (or eventually spiral)
# Return focus to initial position
if style == 'raster': if style == 'raster':
# Return focus to initial position next_z = initial_z
logging.debug("Returning to initial z position") logging.debug("Returning to initial z position")
self.microscope.stage.move_abs([line[0][0], line[0][1], initial_position[2]]) self.microscope.stage.move_abs([line[0][0], line[0][1], next_z]) #RWB: I think this line is redundant
for x_y in line: for x_y in line:
current_position = self.microscope.stage.position
# Move to new grid position without changing z # Move to new grid position without changing z
logging.debug("Moving to step {}".format([x_y[0], x_y[1], current_position[2]])) logging.debug("Moving to step {}".format([x_y[0], x_y[1], next_z]))
self.microscope.stage.move_abs([x_y[0], x_y[1], current_position[2]]) self.microscope.stage.move_abs([x_y[0], x_y[1], next_z])
# Refocus # Refocus
if autofocus_enabled: if autofocus_enabled:
# TODO: Better autofocus if fast_autofocus:
logging.debug("Running autofocus") # TODO: put this in the fast autofocus plugin!
self.microscope.plugin.default_autofocus.autofocus( with self.microscope.plugin.default_fast_autofocus.monitor_sharpness() as m:
range(-3 * autofocus_dz, 4 * autofocus_dz, autofocus_dz)) df = autofocus_dz
logging.debug("Finished autofocus") # move down
time.sleep(1) # TODO: Remove i, z = m.focus_rel(-df)
# now inspect where the sharpest point is, and estimate the sharpness
# (JPEG size) that we should find at the start of the Z stack
jt, jz, js = m.move_data(i)
best_z = jz[np.argmax(js)]
target_s = np.interp([best_z+z_stack_dz/2.0], jz[::-1], js[::-1]) #NB jz is decreasing
# now move to the start of the z stack
i, z = m.focus_rel(best_z - z + z_stack_dz/2.0 + sweep_to_scan_offset) # takes us to the start of the stack
# We've deliberately undershot - figure out how much further we should move based on the curve
current_js = m.jpeg_size()
imax = np.argmax(js) # we want to crop out just the bit below the peak
js = js[imax:] # NB z is in DECREASING order
jz = jz[imax:]
inow = np.argmax(js < current_js) # use the curve we recorded to estimate our position
# TODO: fancy interpolation stuff
# So, the Z position corresponding to our current sharpness value is zs[inow]
# That means we should move forwards, by best_z - zs[inow]
correction_move = best_z - jz[inow] - z_stack_dz/2.0
logging.debug("Fast autofocus scan: correcting backlash by moving {} steps".format(correction_move))
m.focus_rel(correction_move)
else:
logging.debug("Running autofocus")
self.microscope.plugin.default_autofocus.autofocus(
range(-3 * autofocus_dz, 4 * autofocus_dz, autofocus_dz))
logging.debug("Finished autofocus")
time.sleep(1) # TODO: Remove
# If we're not doing a z-stack, just capture # If we're not doing a z-stack, just capture
if (grid[2] <= 1): if (grid[2] <= 1):
@ -172,13 +214,20 @@ class ScanPlugin(MicroscopePlugin):
scan_id=scan_id, scan_id=scan_id,
step_size=step_size[2], step_size=step_size[2],
steps=grid[2], steps=grid[2],
center=True, center=not fast_autofocus, # fast_autofocus does this for us!
return_to_start=not fast_autofocus,
use_video_port=use_video_port, use_video_port=use_video_port,
resize=resize, resize=resize,
bayer=bayer, bayer=bayer,
metadata=metadata, metadata=metadata,
tags=tags tags=tags
) )
# Make sure we use our current best estimate of focus (i.e. the current position) next point
next_z = self.microscope.stage.position[2]
if fast_autofocus:
next_z += autofocus_dz/2 # Fast autofocus requires us to start at the top of the range
if grid[2] > 1:
next_z -= int(grid[2]/2.0*step_size[2]) # Z stacking means we're higher up to start with
logging.debug("Returning to {}".format(initial_position)) logging.debug("Returning to {}".format(initial_position))
self.microscope.stage.move_abs(initial_position) self.microscope.stage.move_abs(initial_position)
@ -190,6 +239,7 @@ class ScanPlugin(MicroscopePlugin):
step_size: int = 100, step_size: int = 100,
steps: int = 5, steps: int = 5,
center: bool = True, center: bool = True,
return_to_start: bool = True,
use_video_port: bool = False, use_video_port: bool = False,
resize: Tuple[int, int] = None, resize: Tuple[int, int] = None,
bayer: bool = False, bayer: bool = False,
@ -211,12 +261,12 @@ class ScanPlugin(MicroscopePlugin):
# Store initial position # Store initial position
initial_position = self.microscope.stage.position initial_position = self.microscope.stage.position
# Move to center scan
if center:
logging.debug("Moving to starting position")
self.microscope.stage.move_rel([0, 0, int((-step_size * steps) / 2)])
with self.microscope.lock: with self.microscope.lock:
# Move to center scan
if center:
logging.debug("Moving to starting position")
self.microscope.stage.move_rel([0, 0, int((-step_size * steps) / 2)])
for i in range(steps): for i in range(steps):
time.sleep(0.1) time.sleep(0.1)
@ -234,6 +284,6 @@ class ScanPlugin(MicroscopePlugin):
if i != steps - 1: if i != steps - 1:
logging.debug("Moving z by {}".format(step_size)) logging.debug("Moving z by {}".format(step_size))
self.microscope.stage.move_rel([0, 0, step_size]) self.microscope.stage.move_rel([0, 0, step_size])
if return_to_start:
logging.debug("Returning to {}".format(initial_position)) logging.debug("Returning to {}".format(initial_position))
self.microscope.stage.move_abs(initial_position) self.microscope.stage.move_abs(initial_position)