background image

27 

NOTICE

: MF can sometimes ‘forget’ the camera is 12-bit (senile code) and the images will come 

up as 8-bit (see scale on side of image or histogram); it’s a bug. This happens if you don’t load a protocol 
before hitting NEW, or actually create a new protocol. To correct it, you need to close the experiment, use 
the File/select video channel/camera and re-select Retiga monochrome-12 bit (even though it looks 
selected). You will be back to normal. So, the first task in creating a new protocol is to go to FILE\select 
camera and re-pick 12-bit mode. 

You should set the sampling time you desire in the Expt Control Panel button ‘Set Timelapse’. 

You can enter the total number of points you desire or the duration. Entering “0” for # points makes the 
collection run until stopped; you still want to tell it how often to sample, though. You can check the 
parameters by using the Acquire One sidemenu button (it grabs ONE SET). The other buttons below it 
grab only the associated wavelength (1-5). 

Be sure to check the boxes for logging ROI data (we’re getting there next) and images otherwise it 

runs and you save nothing. The first time you check a box, MF will ask you for a filename and location. 
Data can be sent to MS Excel and/of a text file. The Save images enables storage of anything (and at 
interval) you set in the ‘Cfg Expt’ menu. The little lights to the right will be colored for data channels that 
will be saved. MF hasn’t evolved from DOS days, so naming schemes still have to be limited to 7 
characters (it appends a ‘d’ to the ‘inf’ name and a 1-5 to describe the wavelength to images, and the file 
descriptor is sequence #)(the inf file contains timing info, event marks, etc; don’t get rid of it). 

Next define ROIs and any 

calibrations and thresholds. Hit the 
Regions button on the sidemenu. The next 
dialog lets you pick the channel to define 
ROIs on and to collect an image if you 
haven’t done that yet. Pick a drawing tool 
from the icons at the top (I like circle), 
start at upper left of where you want to 
draw and you can stretch the size by 
holding left mouse button and dragging 
down and right. You can right-click over 
ROI icons to set default size (make this 
smaller than you normally want, maybe 5-
10 pixels, since it’s easy to stretch it 
during creation). The closed, squiggly 
icon is the freehand draw; you can draw 
while holding the left mouse button down 
(continuously) or tack down a few points 
to have linear spans connecting the ‘dots’. 
You need to double click to stop the ROI. 
ROI can overlap. You can save the ROI 

locations for reuse, else the location of the upper left point is stored in data logs (but no shape/size info). 

Set thresholds on one or more channels to exclude background from ratio calculations. Only data 

above (each) threshold are used in calculations. Only data that are below the top limit are used as well (so 
saturated pixels drop out giving wrong impression that average ratio is dropping; a good reason to save 
images and check for this). Press the 3bar icon, pick autothreshold for light objects and adjust blue 
triangles, if needed, to delimit “signal”. 

Zero the elapsed time clock (Expt Cntl Panel), then hit Acquire or the F2 key (F4 pauses). 

 

ROI 

CLEAR all 

PDF Created with deskPDF PDF Writer - Trial :: http://www.docudesk.com

Summary of Contents for IX70

Page 1: ...nd MetaMorph MetaFluor software to control the system Examples of types of images that can be captured range from simple image capture bright field or fluorescence sequential capture of up to 4 fluore...

Page 2: ...edule the last person MUST shut down the system If there is another user after you 1 Clean any oil or water immersion objectives you used during your session 2 Log off from your Windows session 3 If y...

Page 3: ...rs and measurements incorrect We now have more objectives than positions in the nosepiece Objectives listed below as Available are not normally kept on the microscope but are available for use just as...

Page 4: ...showing nothing of the portions above that zone The high NA yields optical sectioning that approaches that on confocal microscopes Available 60x 1 2 water Use a hanging drop of water not oil on this o...

Page 5: ...he objective in use Due to space cost limitations we have only 3 upper prisms 10x use for 10x 40x oil use for 20x 40x and 100x and 60x TIRF oil use with 60x lenses The prisms match the objectives they...

Page 6: ...exciters or barrier filters to be set by the filter wheels Sutter controller through the software These filters also work by both by eye or by camera However the new quad mirror requires the filter to...

Page 7: ...capture but can be used in custom filter designs as well Need special filters Ask the staff about them we have more filters than turret positions so CFP YFP Chameleon FRET FURA TexRed and Cy7 cubes ar...

Page 8: ...se for either oculars or camera Pushing one of these buttons will pop up a window to tell you which cube to position in the bottom filter turret and then Continuous on the pop up window will open the...

Page 9: ...data itself Images will not save with these settings You can use the GAMMA slider to nonlinearly emphasize the bright or dimmer parts of the image Using a gamma 1 3 or so tends to make the data look...

Page 10: ...ation can be 90 200nm thick a region that limits your view to just the cellular contact to the glass It allows you to view the cellular footprint and can be used to obtain high speed images of changes...

Page 11: ...icrometer on TIRF arm and start image acquisition Note the illumination is constant unless you switch back to mercury arc via the slider or use the manual shutter on the filter cube turret There is no...

Page 12: ...menu bar to generate a live image stream and or slow the data collection rate i e collect an image every second minute hour etc TIRF Shutdown The laser should be turned off by rotating the key to off...

Page 13: ...prefer Major pulldown menus are located in the top bar and organized to task A variety of toolbars appear beside below the main menu You can move the toolbars along the top of the window by dragging t...

Page 14: ...Select OBJECTIVE narrow green Repeated shutter control prompt user to change lens Brightfield lamp ON and load system calibrations REQUIRES filter exchange Close without asking saving preset for 5 col...

Page 15: ...or the floppy disk icon equivalents WARNING keeping any ROI active an active ROI has a hatched appearance will limit all operations to the area inside the region selected that includes what gets saved...

Page 16: ...ons looping and speed controls delete a plane at a time set Z calibration note XY scaling is under Measure menu keep delete range of planes Caution always clear the old ranges before operations it rem...

Page 17: ...coarse or fine select another value from the pulldown list under the SPECIAL tab You can use this menu to set the limits for a z series collection via the Acquire AcquireZseries menu The Apps AcquireM...

Page 18: ...RatioImages creates MetaFluor like output FlattenBackground corrects for uneven lighting when no real background image exists CorrectShading flattens illumination defects based on a bright reference...

Page 19: ...calc objects that are touching w o split IMA sophisticated measure count tool more later Use to define objects in objects Active display of active ROI or window stats Measure one all ROI in one all p...

Page 20: ...matically tracks measures objects speed angles location etc Graph intensities klutzy tool to plot 1 ROI in stacks or live images obvious Window submenus All open windows are listed in LEFT Column can...

Page 21: ...eries MDA main Be sure to select YOUR directory and a name images automatically save there MDA setup Timelapse can enter 0 for number of timepoints to run indefinitely until stopped manually set wavel...

Page 22: ...Click data to show piecemeal or rightclick row or column number to do all each or RtClk upper Left corner for all Select channels to view Check for color overlay or else you will get separate stacks...

Page 23: ...t LoadImage s to extract data into regular image stacks for analysis etc You can make Z projections and rotations with appropriate data input PDF Created with deskPDF PDF Writer Trial http www docudes...

Page 24: ...ratios though most applications would just run two wavelengths Launch using the MetaFluor icon in the Meta Imaging Folder on the desktop or in Program Menu Use the left menu to install a protocol a s...

Page 25: ...ning to decrease image size and increase sensitivity a must for live cells 2x2 binning increases sensitivity of the camera 4X 4x4 increases it 16X I recommend 4x4 and use either all or at least the ND...

Page 26: ...velength save intervals and screen update intervals To run fast you might display a slow step every 3 image To run even faster takes about 50msec to change filter positions you can collect one of the...

Page 27: ...he inf name and a 1 5 to describe the wavelength to images and the file descriptor is sequence the inf file contains timing info event marks etc don t get rid of it Next define ROIs and any calibratio...

Page 28: ...ocus drift bleaching etc You get ratio plots straight away You need to calibrate values to get the real time calibrated value graph to work Calibrate the system if desired using defined buffered solut...

Page 29: ...round using the Acquire button IMAGE mode You can also use a ROI as background if drawn changing the pulldown list This is useful if drugs might alter background during an experiment Flat field or SHA...

Page 30: ...earance not values You can display fixed ranges or autoscale each wavelength using Scale16 The Ratio appearances set using ranges in ImageDispalyControls MF doesn t have the ability to show true ratio...

Page 31: ...ng the dish will increase the rate of evaporation a headache for long timelapse studies If you are doing DIC imaging you need to avoid plastic since it degrades the DIC effect One solution is to use a...

Reviews: