Olympus Fluoview-1000 Скачать руководство пользователя страница 37 | Manualshive

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Olympus Fluoview-1000 Скачать руководство пользователя страница 37

resolved by that objective. Using a zoom setting to create lateral resolution in beyond Nyquist
will not improve the resolution of a confocal image. However, the exception is collecting images
at 2X Nyquist, or 4.6 times the objective resolution, can be useful for deconvolution.

6.11 Obtaining a Z-Series.

The axial resolution for an objective lens is generally 2 to 5 times poorer than the lateral resolution due
to spherical aberrations in the optical path and sample. The sample must be oversampled in the z-axis in
order to achieve maximal resolution, just is is required in the lateral axes. The Fluoview software
calculates the optimal z-step for each combination of objective and wavelength.

The focus steps for objectives in the table below provide sampling to match the Nyquist theorem for
resolution. Smaller values will result in oversampling that may increase detectible resolution at the
expense of inflated file sizes and additional photobleaching.

Table 4. Optimal Z-Step Values

Objective Mag.

Resolution (um)

Lateral Axial

Resolution (um)

Lateral Axial

Optimal Sampling (um)

Pixel Size Z-Step

Optimal Sampling (um)

Pixel Size Z-Step

4X

1.504

54.258

0.654

23.6

10X/.40

0.601

8.56

0.261

3.72

20X/.75

0.319

2.12

0.139

0.921

40X/1.30 Oil

0.181

0.948

0.079

0.412

60X/1.35 Oil
100X/1.4 Oil

0.168

0.778

0.073

0.338

60X/1.20 Water

The resolutions calculated by the Fluoview software tend to be somewhat pessimistic. Axial resolution
is improved by reducing the z-step to 1/2 of these values.

6.12 Resolution.

The lateral and axial resolution limits of the objective lens is set by its NA, not by its magnification.
The resolution limits for any lens are constrained by wavelength, specimen preparation, signal intensity,
noise and sampling frequency. Sampling and resolution on the confocal are properties of zoom and box
size. Most biological specimens do not possess optical properties

Resolution can be defined in 2 ways: 1) How much can you resolve with any given objective lens, 2)
How much resolution do you need to answer your questions? If you are looking for cells that can be
identified by labeling in nuclei that are 5

µ

m in diameter, then you do not need the same resolution as if

you are looking for synapses.

Olympus Fluoview-1000 User’s Guide

V.M. Bloedel Hearing Research Center, Core for Communication Research

Center on Human Development and Disability, Digital Microscopy Center

May 11, 2011

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Содержание Fluoview-1000

Страница 1: ...7 4 15 Line Settings Figure 7 5 15 Scan Controls 16 Scan Mode Figure 6 1 16 Dwell Time Figure 6 2 16 Scan Regions Figure 6 3 17 Image Aspect Ratio Figure 6 4 17 Box Size 6 5 17 Box Size effect on res...

Страница 2: ...Macro 28 Image 5D plugin 28 File Import from the OIF Files directory 29 File Transfer 29 File transfer to Mac Pro Fluoview confocal file server 29 Saving files directly to Mac Pro Fluoview 29 USB and...

Страница 3: ...el Size m versus Box Size and Magnification at Zoom 1 39 Microscope controls 41 Focus 41 Fine Coarse Focus 41 Lowering the Objectives 41 Manual shutter 41 Differential Interference Contrast DIC 41 DIC...

Страница 4: ...eries Control Window 24 Figure 11 Z Series Control Window 24 Figure 12 DIC and Focus Controls Lower Half of the Microscope 40 Figure 13 Condenser and DIC Controls 42 Figure 14 Tilting the condenser 43...

Страница 5: ...he previous user B Shut down 1 Transfer files from your account See File Transfer Section 2 Exit from Fluoview 3 Ignore the warning about light coming through the microscope 4 Turn off power strip B 5...

Страница 6: ...is usually better than brightfield for low contrast samples such as monolayers or picking out structures in complex specimens 5 Epi fluorescence is usually faster than confocal to find the fluorescent...

Страница 7: ...touch Fig 3 1 14 Select mirrors fluorescence filter cubes Microscope Controller Window Mirror click on desired mirror Fig 3 3 see Table 1 15 Fine focus coarse focus press the F C button below each fo...

Страница 8: ...ansmitted light with the Image Acquisition Control Window Figure 4 1 10 Switch to fluorescent imaging Remove the DIC analyzer from the light path for optimal confocal images Figure 2 Enlarged view of...

Страница 9: ...switch to the last filter used 3 Select fluorescence filter if desired a Microscope Controller Window Mirror click on DAPI FITC or TRITC blue green or red fluorescence Fig 3 3 Table 1 4 Adjust focus a...

Страница 10: ...nel is displayed in a separate window 4 Set to gray scale control h 5 Set scanning to simultaneous Sequential checkbox Fig 4 9 is cleared 6 Initiate scanning Image Acquisition Control Window Focus X2...

Страница 11: ...ctions b double click on any selected dye to remove it 3 Scroll through the list of dyes in the lower window 4 Select each desired fluorophore by double clicking on the name a if your particular dye d...

Страница 12: ...ing the stage b may be used for recording rapid fluorescence changes such as ion measurements 2 Focus X4 4 5 Scans alternate odd lines replicating the scanned lines to generate an image of 4X reduced...

Страница 13: ...ile this can provide brighter images the noise and background intensities are increased as well a leave set to 1 unless working with very low HV settings 3 Offset 5 3 The Offset will raise or lower al...

Страница 14: ...a check mark Figure 7 1 Clicking in these boxes will toggle lines between active and blocked This control blanks the lines in the scanhead it does not turn the laser itself on or off 2 5 2 Line availa...

Страница 15: ...he slider 2 5 5 Line Settings Figure 7 5 As with other controls the laser power may be adjusted by clicking anywhere in the control area scrolling the thumbwheel 1 increments or by clicking on the bla...

Страница 16: ...ent moving over each point to deliver more photons exciting the fluorescent label to generate signal However a longer dwell time will increase photobleaching The time required to scan a field is deter...

Страница 17: ...Normal rectangle defines the scanned region as the selected box size The Polygon options allows user defined regions within the box These may be rectangular circular or irregular polygons The laser be...

Страница 18: ...dwell time may cause your image to become saturated 1 Begin scanning with Focus X2 or Focus X4 Focus mode and find your sample 2 Adjust the laser and detector settings 3 Click on Auto HV to set the re...

Страница 19: ...plished by clicking on the Zoom Reset then changing the zoom to the new value 2 7 6 Scan Offset 8 6 Move the Scan Area within the Field of View by either clicking on the scan area and dragging it or b...

Страница 20: ...ng Controls 9 1 Sequential Line vs Frame 9 3 Channels 9 3 Kalman Averaging 9 2 Groups 9 2A 9 3A Grouped Channels Olympus Fluoview 1000 User s Guide V M Bloedel Hearing Research Center Core for Communi...

Страница 21: ...nels If a laser line also significantly excites other fluorophores their emissions may overlap sufficiently to cause bleedthrough As always control specimens are important Simultaneous is faster than...

Страница 22: ...g 1 Live Window is set to gray scale control h in order to observe saturated pixels 2 Set Live Window for tile view each detector is viewed in its own pane 3 Click on Sequential 4 Select Frame 5 Click...

Страница 23: ...collected with an increment of focus between each image plane This is an automated process where the user sets the upper and lower limits of the focus travel with an interval for incrementing the focu...

Страница 24: ...End 10 5 Set Focus to 0 11 2 Optimal z Step 11 4 of Slices 11 3 Focus Increment 11 5 Clear Start End 11 1 Set Focus Limits 11 7 Step the Center of Z Series 11 6 Step Focus Full Step 1 2 Step Olympus F...

Страница 25: ...append is controlled by the box next to the button The red number below the lens icon in this window displays the current axial position You can simplify keeping track of focus distance by clicking o...

Страница 26: ...gions of interest and preserve the original 12 bit 4096 image intensity values Each format has its benefits and drawbacks 5 1 1 The Olympus OIB format This format writes all images and meta data to a...

Страница 27: ...the scale bar show up in ImageJ or Photoshop However this means saving your file under yet another format Adding scale bars in ImageJ or Photoshop is simple and directions can be found on our website...

Страница 28: ...Olympus datasets into ImageJ 5 4 1 Loci tools Plugin for ImageJ The LOCI plugin will import about 80 file formats including OIB and OIF You will be presented with choices for merging or splitting cha...

Страница 29: ...iles shortcut on the confocal desktop 4 Navigate to your user account and the Files directory located in Drive D 5 Drag your confocal files from the User files directory to your directory in the Mac P...

Страница 30: ...e list of favorites b type in your server address name or TCP IP address c Browse the network 5 5 6 Using FTP to Windows computers 1 FTP transfer to your server from Fluoview Mac Pro by the Fetch appl...

Страница 31: ...aching 3 Channel Offset should be adjusted to remove blue pixels usually 5 to 7 4 HV and laser power levels should avoid saturation red mask 5 Establish imaging conditions using monochrome display rat...

Страница 32: ...bodies subjected to immuno absorbtion are also useful 6 3 2 Positive controls Known positive controls are also important Positive controls labeled for each single label are crucial when imaging specim...

Страница 33: ...he channel s in which the bleedthrough is observed High excitation intensity will cause the low probability tails of an emission spectrum to reach detectable levels in adjacent channels for most combi...

Страница 34: ...s by the square root of the number photons from the mean of the population This variation represents 1 standard deviation on each side of the mean number of photons This translates into the reality th...

Страница 35: ...nsities that can not be attributed to specific or desired label signal Background reduces contrast and dynamic range interferes with computerized feature recognition and image processing Sources of ba...

Страница 36: ...r spot and in doing so changes the sampling rate for the image Zoom provides additional resolution without the need to increase magnification as with a widefield microscope and camera 6 10 1 Zoom redu...

Страница 37: ...Optimal Sampling um Pixel Size Z Step 4X 1 504 54 258 0 654 23 6 10X 40 0 601 8 56 0 261 3 72 20X 75 0 319 2 12 0 139 0 921 40X 1 30 Oil 0 181 0 948 0 079 0 412 60X 1 35 Oil 100X 1 4 Oil 0 168 0 778...

Страница 38: ...2 Box size This term refers to the number of sampling points in the scanned field A box size of 512x512 means that the field will be divided into 512 samples along each of 512 lines If the lines in t...

Страница 39: ...0 132 2048x2048 0 621 0 310 0 155 0 103 4096x4096 0 310 0 155 0 103 0 051 The size of a pixel in a zoomed image is obtained by dividing the value from the above table by the zoom factor Shifting from...

Страница 40: ...or for 2X oversampling Pixel size 2 wavelength NA Figure 12 DIC and Focus Controls Lower Half of the Microscope 12 1 Focus Knob 12 4 DIC Analyzer slider out position 12 3 Lens Drop Raise 12 6 DIC Anal...

Страница 41: ...rference Contrast DIC This enables high contrast high resolution images by transmitted light from samples that are only stained with fluorescent labels Using DIC greatly reduces photobleaching while l...

Страница 42: ...ton prism into place 7 3 Setting Koehler Illumination Maximal resolution and field uniformity with brightfield and DIC is only achieved if the condenser is in focus to the same plane as the objective...

Страница 43: ...ht intensity is restored 9 Check the focus of the sample it may change if the condenser was initially out of focus 10 If the focus was changed re check the condenser by simply closing the field stop a...

Страница 44: ...om to a value greater than Zoom 1 concentrates the laser power of into a much smaller volume The result is that the photodynamic effects are achieved with much lower laser level and shorter exposure d...

Страница 45: ...umber of scans or a defined time period ms f Activation in Series allows photoactivation operations in a sequence i PreActivation record a specified number of images before activation ii Activation Ti...

Страница 46: ...cence to confocal imaging Rotating the mirrors filter cubes to allow confocal scanning exposes the entire field of view to a brief flash of UV from the DAPI cube Close the manual shutter below the obj...

Страница 47: ...em redo work space color Figure 17 2 4 Be aware that menus and toolbar items are nearly impossible to see in Darkroom color mode 17 1 Darkroom Color 17 2 Redo Work Space Figure 17 The toolbar item for...

Страница 48: ...s e g Cargille Type B for the least spread of oil 8 Do not over oil immersion objectives use the minimum amount of oil necessary a Scrunchies have been wrapped around the oil immersion objectives to a...

Страница 49: ...hrough dichroics measured at the lens turret 100 output Dichroic Mirror 405 nm 458 nm 488 nm 515 nm 561 nm 633 nm BS20 80 161 W 9 4 W 23 5 W 50 W 45 W 98 W BM405 488 414 W 1 W 102 W 16 W 1 2 W 20 4 W...

Страница 50: ...ncreased Has the detector Offset been increased This should be 5 7 Are you adjusting to the correct PMT and laser Does the Confocal Aperture need to be opened up Do you need to use special imaging met...

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