Guide 1. User Guide. window. program.

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1 Guide 1 Olympus BX53 Digital Mirosope User Guide A. System Start up. 1. Sign into the log ook on the desk (start/end time). 2. Turn on (or wake up) the omputer and selet USER and enter the password on the log in window. 3. Turn on the mirosope, the main ontrol unit, the Prior remote z stage ontrollerd, DP26 olor amerae, andd Xenon lampf (only if you need it for fluoresene imaging). 4. Start ellsens Dimension program. B. Hardware Control and Setting. 1. Light path arsg direts light to eyepiee, amera, or oth. 2. Selet an ojetive lens y turning the nosepiee turreth y hand. 3. Halogen lamp dial kno iontrols the rightness of transmissionn light for right field viewing. The transmission g e g d h f i j light will e visile from the ondenser lens. 4. Prior Z stage Control Boxdd moves the stage up or down to fouss on speimen. The UP utton rings the stage up lose to the ojetive and DOWN utton rings it down quikly. Finer movement of stage an e ontrolled with the kno; the extent of the fine movement an e seleted High (H), Medium (M), or low (L) y pressing SPEED utton. The seleted speed appears on the LED sreen (Note:: DON T USE the fous kno on the mirosope and the ZERO utton on the ontroller.)

2 Guide 2 5. Remote ontroller oxj ontrols the seletion of imaging mode (i.e. BF or Fluoresene (FL)). Use the Mirror uttons to selet an appropriate filter mirror ue for different olors of fluoresene. Press the FL Shutter utton too open or lose the merury ar lamp light shutter. C. Overview of ellsense program and asi image aquisition. 1. When the program opens, first set up the program for your imaging needs (i.e. right field imaging for histohemially stained samples or fluoresene imaging for fluoresently laeled samples). Clik the Aquisition window ta. 1) 1! 2. Selet an ojetive (start with 5x or 10x ojetive) y pressing an ojetive utton. Thee stage will lower and a message windoww will pop up. Turn the ojetive nosepiee turrethh y hand to put the mathing ojetive in plae. Clik OK on the message window (Itt is ritial to math the magnifiation with the ojetive to otain orret sale ar). 3. Clik one of uttons on the Oservation Methods panel for rightfield or fluoresene viewing. 4. Fous onto the sample y moving stage with Prior z stage ontroller or y liking arrowheads in the Devie Units panel.

3 Guide 3 5. Selet a amera from the Camera list1) ): XM10 monohrome amera for fluoresene or DP26 olor amera for right field. Selet Manual or Automati exposure to otain etter images. With DP26 amera seleted, selet Automati from the white alane mode list. 6. Selet Resolution1! as desired. (Note: You an hoose a lower resolution for Live/Movie for fast update of live (ative) sene.) D. Bright field (or DIC) Image Aquisition 1. Make sure that DP26 amera is ON and seleted from the ameraa list and that the upper light path is pushed in and the lower ar11# is pulled out. 2. Clik Live utton and otain proper exposure and whitee alane ( Automati exposure1$ with Spot 30% region and automati white alane1% % work fine for most ases). Clik the utton again to stop live viewing. 1# 1@ 3. When the setting is satisfatory, lik Snap utton and save the image as.vsi or.tif format y liking the right mouse utton over the image ta. 1$ 1%

4 Guide 4 E. Fluoresene Image Aquisition 1. Clik the FL utton on the remote ontroller ox to use merury ar lamp light for fluoresene. The light an e on or off y pressing FL Shutter utton on the remote ox. d e f 2. Selet an appropriate fluoresene mirror ue y pressing Mirror uttonsd in the ontroller ox or liking any one of the Oservation Methods uttons (exept BF and DIC). The seleted mirror will e in positione. Make sure that the light path arsf are in orret position (the upper ar at the mid or extended position and the lower ar pushed in) and XM10 amera is seleted.

5 Guide 5 3. Let s take multiolor fluoresene image aquisition with an example stained with DAPI, FITC, and TxRed fluorophores. 4. Clik the Proess Manager taf and hek the Automati Proesses g. Clikk the multiolor wheel utton. Clik the uttonn and selet DAPI (lue) in the list. Repeat twie to selet FITC (green) and TxRed (red) hannels. Clik the plus sign next to the amera ion; it will expand to show urrent exposure setting. Clik the Live utton. If Automati exposure (with Spot 30%) is used, let the program find the appropriate g exposure time. One the exposure onditions are set, lik ion to set these exposure onditions for DAPI. 5. Clik the FITC setion on the list and use the Automati exposure. Wait until it finds an optimal exposure time and lik ion to read the setting. Repeat one more for TxRED hannel. One all the settings are otained, lik the utton. It will take three shots for eah hannel automatially. The resulting multihannel image, y default, will e saved as.vsi file in the Liraries>Pitures folder as soon as the aquisition has een ompleted. Save the original image files as.vsi format without any manipulation or proessing. 6. Images an also e saved as.tiff files. Selet Image>Mode>8 it/channel menu ommand to hange the image into 8 it image. Use File>Save as menu ommand to save the image as one image ontaining multiple hannels. Or use File>Export to> >tiff series ommand to save the image as multiple images for eah hannel. 7. Sale ar an e viewed with View/Salee ar ommand and the magnifiation info an e displayed with View/Info Stamp ommand. These annotations an t e saved with your tifff image. Save the annotated image as JPEG(JFIF)(*.jpg). f

6 Guide 6 F. Image Proessing 1. One you save the original file, make a dupliate if it is neessary to make any hanges in the image for presentation or puliation. Clik the Proessing window ta from the main window tas, in whih the image histogram and the rightness and ontrast of the display an e adjusted. 2. In default, the adjustment of the Histogram or the Display Enhanement applies to all the visile hannels. On the Dimension Seletor panel, you an hoose the hannels to e visile (eye symol indiates visile and grey to hide out eye symol indiates invisile ). Clik the olor hannels you want to show and lik on the eye symols unwanted hannels. Then, you an adjust the histogram and the rightness/ontrast of the seleted hannel only.

7 Guide 7 3. To save some overlaid images from a multihannel image you justt made as aove, there are three ways (note: these operations willl automatially hange the image type from 16 it graysale to 8 it olor): ( 1) Make a omposite image as aove. From the Edit menu, use Edit/Selet All, then Edit/ /Copy, and then Edit/Paste As/New Image. This operation will generate a omposite image with the same sale ar. ( 2) Make a omposite image as aove. Use Edit/Copy Display to o Clipoard and then Edit/ /Paste As/New Image. This will reate a new image with sale ar in pixel unit (losing some dimensional information). ( 3) Selet all hannels from the Dimension Seletor. Use Image/ /Separate/Channels ommand to separate the multihannel image into individual hannel images. Then usee Image/Comine Channels. In the window, selet hannels to e omined and lik OK.

8 Guide 8 F. Aquisition of Z stak (z series) 1. A z stak image ontains multiple frames aquired at different fous positions. Clik z stak utton to display Z stak panel. To set the Start and End limits of a z stak, move the stage using the Prior stage ontroller or y liking the arrowheads in the Proess Manager window. First, lower the stage to fous onto a region of interest lose to the over slip and then lik Set utton, whih will e the End position. Next, ring up the stage to fous onto a deeper region lose to the slide and lik Sett uttond, whih will the Start position. (Image aquisition will egin at the Start position.) Ojetivee d over slip Slide End Start on stage e 2. The program will automatially reommend a step size efor z series aording to the f ojetive. Clik Apply utton to aept this step size or hange as you want and apply. It will alulate the numer of z sliesf aordingly. 3. Clik the Start utton to apture a z series. You an omine this funtion with multiolor imaging. Save the images. G. Displaying and Converting z staks 1. In the Proessing ta, the z stak image an e manipulated in various ways to generate different views. 2. With the navigator ar frames of a z stak. 3. You an selet a single frame from a z stak y liking the right mouse utton in a single frame view and seleting Extrat Currentt Frame from the ontext menu. 4. You an also selet a short range of frames within the stak y using Set Seletion uttonsg; determine a new starting position within the stak andd lik Set Seletion Starting Position and hoose a new end position and lik Set Seletion End Position. Then hoose Tile View. Make sure all the hannels are visile (seleted). Over an image, lik the right mouse utton and selet Extrat from the ontext menu. A new z stak ontaining only seleted frame will e generated as a.vsi file. Savee the image., you an navigate different foal g

9 Guide 9 5. You an projet all the z stak frames into a single 2D image y seleting Proess/Projetions/Maximumm Z ommand from the main menu or Maximum Projetion Over Z on the navigator ar menu. 6. With the Slie View menu, you an display a ross setional z dimension view of an x and y plane. You an view anywhere y moving the xy ross hairline to the desired position. 7. With the slie view menu options dimension view of extendedd x y slie position., suh as Extended Slie, you an visualize z

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