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will allow even red probes to be used provided they can be excited at 488nm (perhaps fura-red or RFP). 
You can narrow the emission by using the 535 barrier filter in the camera filter changer (FITC (535BP) 
TIRF
 filter definition in taskbar menu). Be aware that CFP will not be excited very well at 488nm (in fact, 
it’s peak emission is near this wavelength). See below for protocol. 

By turning the micrometer CCW you are 

marching the beam downwards (toward the periphery of the objective back aperture), and when the external beam disappears, 
you have reached the critical angle for TIRF. It’s convenient to set the depth of the TIRF illumination while observing the live 
image on the monitor.

 You may wish to use a camera sub-region, bin pixels, or both, to get fast image capture 

rates. The frame capture rate can be read off the Acquire window 
under the Live Bin box.  Fast acquisitions may require using the 
“stream acquisition” feature (Acquire 

 Stream Acquisition) 

where data is grabbed as fast as possible without any active 
display to slow things down.  

By pulling up the illumination path slider (behind the 

scope) up, you can switch to the conventional mercury arc 
illumination to capture a standard image. Be sure to use the 
software TIRF illumination setting since this places the excitation 
filter into the path (as well) and prevents you from blasting your 
sample. Push the slider back down to return to TIRF mode. 

 
HOW-TO TIRF: Use the Plexiglas reflection shield (fix on scope just behind the eyepiece 

mount) Watch out for beam reflections! This is more likely if you use a metal chamber. Protect other 
users from stray beams as well. Never stare at a laser beam. The beam is low intensity, but eyesight is 
never worth even a small risk. 

The procedure is to first find your candidate cell using regular fluorescence (plunger slide UP 

behind the scope), then switch to camera, live view, TIRF illumination (plunger slider DOWN behind the 
scope), adjust the field depth of illumination (micrometer 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 automatic shutter for laser light (yet). TIRF is generally far 
less toxic and bleaches less than regular illumination. You can insert a ND filter with the slider on the 
TIRF tube (see left image on pg. 10) to decrease TIRF laser intensity, if needed. Power can also be 
modified to some extent by the trim pot on the front of the laser power supply if you use the non-standby 
mode. Generally there is more than enough light in standby mode for GFP work in shallow illumination. 
DsRed (and other RFPs) can be excited by 488 and you can use the colored filter to restrict capture to 
only the red component if desired. You can sequentially collect the green-only and red fluorescence by 
setting a protocol to switch the emission filter wheel using Multi-dimension Acquisition sub-application 
(see below). The filter can change in about 55 msec. Alternatively, you can use the image splitter tube and 
the split image capture mode in MetaMorph to simultaneously grab green and red signals, although the 
region you can capture will be smaller. 

Check the setup with ShowLive. How fast is it going? Check the Acquire/Acquire menu bottom 

left side (in show live mode) for the frames per second (fps). To capture data use the 
Acquire/StreamAcquisition menu item (this stays open) and stream to memory or disk. It will warn you if 
the requested data won’t fit into memory (but only when you actually try to collect it). Note that the 
stream mode does NOT give a live image. You won’t see what’s happening while streaming (it’s 
designed to go as fast as possible) until you play back the stream. Don’t collect full images (approx 1.4 
MB each) when your target is only a tiny fraction of the image. Draw a box around the bit you are 
interested in, and then use the Acquire/Acquire/Use Active Region button to keep only the important bits. 
This saves memory and allows for faster capture (less to read back). The Center Quadrant button may be 

 

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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...

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