Olympus IX70 Betriebshandbuch - Seite 8

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fluorophores, you can make the task easier by using the Acquire Multiple Wavelengths menu. This

automates collection of up to 6 "channels" of data, each stored with a fixed/different exposure (or

autoexposure). To use the Acquire multiple wavelengths menu, pick the number of probes to collect and

assign the identity and order in the lower section. Pressing the upper right camera icon collects the entire

sequence and stores it in one memory stack. For this to work, the microscope has to be set up with the

quad mirror in place in the lower filter turret. If you want to use the individual color cubes and change

cubes between captures, pressing the cameras next to each probe collects only that probe. To collect color

(histology) images, make sure the order is colorRed. colorGRN, colorBLU. Gain is assigned from the

main "acquire" window and is applied to all channels. Independent exposure times can be assigned by the

user or calculated for every capture by checking the "autoexpose" box. Autoexposure takes time since a

series of exposures are tried and evaluated. You may want to disable the auto feature after the first pass

and used fixed times thereafter. You certainly do NOT want to autoexpose everything if you doing a study

buttons. The lefthand side is all buttons for the single color cubes (use 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 shutter. You will be able to scan for

your sample with one of these cubes, but be sure the light path is set to EYE (check that the knob on the

right-hand side of the scope is set to the eye image). If it set to camera (SP, or the icon for the non-existent

manual camera) rotate the wheel until it clicks and you see the eye. Focus upwards (CCW, 'fingertips up

= up') to find your samples, but keep in mind there is NO limit in place to prevent you from crashing into

things and breaking your prep, or worse, an objective. If you are having trouble here, ask for help.

Unstained cells may be easier to find with DIC, but I prefer to use fluorescence mode for stained

materials. If you have a DAPI stain, this works particularly well.

3) Find the cell(s) you wish to image and place them just to the right of center. The camera sees roughly

the center/right 50% of what you see. You should then verify that the camera sees the same focus that you

did by pressing the "Show Live" taskbar button (middle left column)—this reminds you to change the

light path by rotating the knob to the "SP" side port setting and then starts the camera+screen display.

You can use the Acquire menu "autoexpose" button to set the brightness (this can take some time with

dim samples), or enter numbers directly. With no binning and gain=1, 100-200 msec should give a decent

image (on average). (exposure testing is set to max out at 10 sec). If there is no image on the screen,

double check that you remembered to send light to the camera.

allow you to change "illuminations" and objectives. Be aware that these menus do LESS than the similar buttons in the taskbar.

Changing a filter, for example, only tells you to change the filter, while the task button would also then open the shutter...

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where staining intensity is important

(e.g., treatment vs control). If you want

to time-lapse this sequence and/or do a z-

axis collection, then use the APPS menu

routine Multi Dimensional Acquisition.

Again, for multiple wavelengths, the

quad mirror cube must be used in the

filter turret.

2) Pick one of the illumination settings

from the taskbar (2-column menu).

There are two columns: the righthand

side is all buttons that work with the

"quad" mirror (camera only). Change

the manual filter turret to position 4 and

flip through the channels with these

Note: There are pulldowns at the top that also