User manual for Nikon Elements software

Equipment: Nikon TE300 Eclipse microscope ANDOR Neo/Zyla B&W camera (default) DS Fi2 color camera Sign in on the sign in sheet; please use both your given name and your surname, your department, your PI, and provide a telephone number at which you can be reached. If you do not use your lab telephone as your main contact, please provide your mobile number.

If the room lights are not on when you arrive to use the Nikon, DO NOT TURN THEM ON. Please be courteous of those using the other microscopes, since they may have light-sensitive slides, dishes, or plates on the microscope stages, which could be damaged by the room lights. This is particularly the case with the Leica confocal, since some Z stacks can take over half an hour to complete.

The default camera attached to the Nikon microscope is the B&W ANDOR Neo/Zyla camera, which is the camera used to capture fluorescent images. It will be attached to the microscope at all times unless removed by me or another member of the RCF staff to attach the color camera.

If you need to use the color camera, please let me know ahead of time so I can remove the ANDOR Neo/Zyla camera (B&W) and install the DS Fi2 color camera. The current policy is that the color camera will only be available for use during regular business hours (M-F, 8:30 am-5:00 pm); any after hours or weekend use must be arranged ahead of time with me.

The light source used to excite fluorescence has been changed from a mercury arc lamp to a lamp with an LED light source, so there are only two things to turn on: the Tripplite Line Stabilizer, on the shelf above the microscope, and the computer itself. The line stabilizer provides power to the microscope, camera and stage, and prevents damage to delicate circuitry from power surges.

Turning on/preparing microscope

Turn on the Tripplite Line Stabilizer with the rocker switch on the lower right Turn on the computer (the button is located about halfway down the length of the computer)

Once the computer is on, please log on to the computer (rather than the network) using the following

user ID and password:

User ID: nikonuser Password: corescopes

Before you open the software, you need to decide which camera you are using, because the camera

must be ON; this allows the software to locate and connect with the correct camera drivers.

If you are using the B&W camera to take images of fluorescence, turn on the ANDOR Neo/Zyla camera

by gently flipping the small switch on the back of the camera (see photo below). It is sensitive, so please

do not exert force.

For the color camera, turn on the Nikon Digital Sight DS-U3 unit, which is connected to the camera via a cable. The green light will come on; once it stops blinking and remains a solid green, the camera is ready to use. Ensure that the small lamp sitting on top of the DS-U3 unit is NOT BLOCKING the air intake. Open the NIS Elements software by double clicking on the icon. In the log-in dialogue box, log in using the same information as above: User ID: nikonuser Password: corescopes In the camera dialogue box, choose either the ANDOR Neo/Zyla camera or the DS Fi2 color camera.

ANDOR Neo/Zyla camera, switched ON Digital Sight DS-U3 unit

The Nikon lamp switch on the left side of the scope should be pushed in (ON). If not, push it in.

Check to see that the ND filter sliders and the fluorescent manual shutter are pushed in

Place your sample securely in the stage tray. Remember to invert slides, coverslip down.

Move the Light Path Selector Wheel located on the right side of the microscope to position A. The wheel will click when it is correctly positioned at A.

The motorized stage joystick located to the right of the microscope has three speeds: 100%, 50% and 25%, which can be changed by pressing the switch to the left of the joystick. The switch continuously cycles through the speeds, so try different speeds to find the one you prefer.

Nikon lamp switch Filter sliders Manual shutter

Motorized stage joystick Light path selector wheel

Setting up for imaging (objective, plates, slides)

Choose the correct stage insert and use the nylon screws to fasten it in place (extra screws in clear

plastic box to right of microscope along with long screwdriver with amber handle).

Choose the objective you wish to use. The objectives currently mounted on the turret comprise the

following: 4x, 10x, 20x, 20x, 40x, 40x oil. Please note that only ONE of these is an oil objective, requiring

the use of immersion fluid; the others are all air objectives, which do not require any immersion fluid,

and will in fact be damaged by oil, glycerin, or water. The turret is rotated by hand; please do not touch

the objectives as you rotate the turret, since those with correction collars can have the collars

inadvertently changed. The objectives are shown graphically in the software in the same order in which

they are found on the turret. If needed, 60x (oil) or 100x (oil) objectives are available to be checked out.

PLEASE use ONLY the Nikon oil in the brown plastic container at the microscope station with the 40X oil

objective.

The Nikon is an inverted microscope, which means that slides should be viewed coverslip DOWN

(closest to the objective), so slides should be flipped over. The microscope objectives are engineered to

work best with coverslips 0.17 mm in thickness, usually referred to as 1.5 or 1 ½ when ordering.

Plates and dishes should not be flipped. If possible, you may want to remove plastic lids, since plastic

can distort imaging.

The coarse focus knob is located on the R side of the microscope. The fine focus knob is located on the

R side of the joystick pad that controls the X-Y movement of the motorized stage.

Filter wheel positions (L to R):

Use filter wheel located inside microscope

NO filters (color camera, B&W + phase)

Red filter cube

Green filter cube

The Filter Wheel Positions Lever controls which filters are in the light path during observation and

imaging. It slides along the bottom of the black box located on the R side of the microscope stand. There

are four positions: all the way to the LEFT (Filter Wheel Positions) tells the software to use filters from

the filter wheel located in the inner back portion of the microscope (DAPI, FITC, TRITC). As the lever is

moved to the right through the next three positions, the software is instructed to use NO filters

(Dummy, for bright field/phase contrast), the Red filter (excitation 545/30x, emission 620/60x), or the

Green filter (excitation 480/40X, emission 535/50x).

Observing images through the eyepieces (light path: A)

When viewing your sample through the eyepieces, set the selector knob on the lower R side of the

scope to A, which directs 100% of the light to the eyepieces. B & D allow light to go to the eyepieces and

the camera at the same time; C directs 100% of the light to the camera.

OC Panel Tab: Filters. Click on the Filter Block graphic that allows you to choose either the internal filter

wheel (for Blue/Green/Red), the neutral position (for bright field, phase, and/or the color camera), the

dedicated bandwidth red filter cube, or the dedicated bandwidth green cube. This corresponds to the

hardware positioned on the right side of the microscope stand.

Filter block (software) Filter block (hardware)

OC Panel Tab: Objectives. Click on the objective you will be using (please note that only 4x-40x are

currently mounted on the turret; please let me know if you need to check out the 60x and/or the 100x.

OC Panel Tab: Filter Cubes. Click on either the cube (DAPI, FITC, TRITC) for fluorescence, or the

Transmitted light (DIA.ILL) for bright field or phase contrast.

Objectives

Filter cubes

Transmitted light

To use bright field illumination, go the tool bar at the top of the software and click on the DIA button.

Unclick the box for the λ tab at the bottom of the page.

For fluorescence, go to the tool bar and click on the SOLA button.

The button marked AUX is an additional shutter operated by the software.

Observing live images on the computer (light path: C)

When viewing the sample in the software, set the selector knob on the lower R side of the scope to C, which directs 100% of the light to the camera. To create a live image, click the green Arrow button in the tool bar at the top of the software. A dialogue box/window will pop up. Use the fine focus knob to adjust the focus on screen. If you are using the color camera, you can also adjust the white areas of the background (AWB at the top of the dialogue box, or the Auto White button in the area on the left).

Green arrow: Live image Red square: Freeze Camera (blue dot): Take snapshot

Setting up channels for imaging (fluorescence)

Go to the ND Acquisition tab (lower left area of screen). You can choose among tabs for T (Time), XY, Z, λ, or Large Image. To set up a multichannel scan, click on the λ tab.

ND Acquisition showing the λ tab with three channels

Ensure that the other tabs are unclicked unless you are using them.

Under Set Up, you will see boxes where channels can be added; press the blue plus sign (+) to add or

the red X (X) to remove channels.

New channels will appear under Optical Conf. Use the drop down menu to the right to choose among objectives, the diastolic shutter (DIA.ILL), or fluorescent filters. Under Name, the software will fill in the name of the tab that appears in the main screen of the Live dialogue box. Under Comp. Color, you can use the drop down menu to choose how each channel appears in the image.

Removing background

There are two ways to remove background fuzziness from fluorescent photos, either individually or, if

required for images in a series, by choosing a set value.

Method 1: Capture an image. On the right side of the image window, locate and click the 2nd icon, which

is the Background ROI button. This will create a rectangular ROI that can be adjusted in size and

positioned over an area of background with a uniform color. Once this has been done, go up to the top

of the software and click on Image. From the drop down menu that appears, click on Background, then

on Background using Background ROI. This will remove scattered light from the background and help

clean up your image.

Method 2: This allows you to choose a constant value that you can apply to images in a series. Follow

the same procedure as described above, except when you click on Background, choose Background

using Constant. You will be able to type a value for the constant into the menu; you can determine the

constant by looking at the values generated by moving the cursor around the background (they will

appear at the bottom of the dialogue window) and deciding upon an average value. This can be applied

to images in a series.

Adjusting lamp intensity

To adjust the lamp intensity, drag the white bar to the left (reduce) or the right (increase) to change the

intensity of the lamp. Click the white arrow to the right of the filter cube you are using to set the value.

Unless you change it again, or close the software, the value you set will remain in place during your

entire session.

Shadowed edges on images

The chip inside the camera is larger than the side camera port on the microscope stand, which results in

differences in the way the field of view appears through the eyepieces and in the Live window. This may

cause a dimming of the signal from fluorescently tagged cells, especially in the corners of the photos. To

minimize this, within the Live window, go to ROI and choose a smaller area.

Creating a merged image

If you have taken images separately (for instance, you need to take both fluorescent and bright

field/phase contrast images, and would like to merge them), you can create a merged image using them

by placing the mouse cursor on the tab of one image, L clicking, and dragging it on top of one of the

other images. The original of the one you dragged will remain; in the window with the image to which

you dragged the first image, you will now have two tabs: one for the new merged image, and one for

the one onto which the other image was dragged. You can do this with multiple images. If you no longer

need the individual channel images once you have made the merged image, you can delete them.

Saving an image (ND, TIFF, JPEG; folders; offline access)

When you are ready to save an image, go to File and choose Save As. You will have choice in which

format to save the image. Each user has a personal folder located in the Users Folder on the desktop.

Images can be saved there temporarily, but should be removed in good time. The easiest way is to use a

flash drive and the USB ports on computer.

The proprietary file format is .ND2, which can only be opened with the NIS Element s software. It is

important to save your original image, because the software stores information about the image that is

useful. In addition, you can always make a .tif or a .jpg file from your original, but you cannot go back.

Please note that when saving as a .tif, it is important to look under the TIF compatibility options and

choose the third radio button: Scale 11 bit to 8 bit. This allows you to open the .tif file using any viewer

you would use to view .tif or .jpg files; the drawback is that it reduces resolution.

Shutting down the equipment

If you have used oil, make sure to clean the objective with the non-abrasive lens paper provided.

Currently, this only applies to the 40x oil objective used for fluorescent imaging.

Save any images that you need to, and take with you (flash drive is the easiest way) any images you

need immediately. Exit the software by clicking the red X in the upper R hand corner of the screen. A

message will pop, saying ________________________, to which you should respond by clicking Yes.

Click on the Start button in the lower L hand corner of the screen and choose Shut Down.

Turn OFF the surge protector on the shelf above the microscope.

Turn off the ANDOR Neo/Zyla camera by GENTLY flipping the switch on the back of the camera to the

right (towards the user), or the DS Fi2 color camera by turning off the Nikon Digital Sight DS-U3 unit.

Replace the cover and remember to sign out.

ANDOR Neo/Zyla (B&W) camera

On the microscope itself, remove the NCB, ND, and/or GFP filters from the light path. The first two

filters remove the yellowish cast that can occur with bright field images, but are not usually necessary

for fluorescent images. The GFP filter creates a strong greenish cast. While these filters do not seem to

change the image (except for the GFP filter), you can try any or all of them with your

slide/plate/flask/dish to check whether using a filter helps.

Push the Filter Wheel Positions lever (located on the right hand side of the microscope stand) into the

first position, labelled Filter Wheel Positions, which is the leftmost position. This lets the software know

that you are going to use the filters on the wheel located inside the microscope (DAPI, FITC, TRITC). If

you want to use either of the other two filter cubes (Red or Green), slide the lever into either of those

positions.

In the software:

Go to the Zyla Settings tab. You will see areas (divided from one another with black lines) along with

buttons and drop down menus to allow you to control how your image is captured and displayed.

Zyla settings tab

Under Format, you can change the binning of the pixels (increases the brightness of your image). The

Binning drop down menu gives you the default choice of No Binning, or 2x2, 3x3, 4x4, or 8x8. You can

also click on the Auto Exposure button to allow the software to choose the length of exposure, or use

the drop down menu to choose the length of exposure manually from among a preset list.

In the next section, you will see:

Readout Mode: set on Rollling shutter(default), with a drop down menu that allows you choose

between Rolling shutter or Global shutter.

Readout Rate: set at the default 560 MHz, with a drop down menu that allows you to choose

between 560 MHz and XXX MHz.

Dynamic Range: with a drop down menu that allows you to choose among 11-bit & Gain 1

(default), 11-bit & Gain 4 (best for fluorescent imaging), or 16-bit & Dual Gain ¼ (the setting

used for Phase Contrast).

Sensor mode: set on Overlap (default), with a drop down menu that allows you to choose

between Overlap and ________.

The greyed out box labelled Limit Maximum FPS to, with a box for typing in a value, shows (to the right)

the value chosen by the software. If you check the box, you can change the value; otherwise, the

software chooses for you.

The box will be checked for the Spurious Noise Filter , which is the default setting.

Go to the Filters, Shutters and Switches tab. You will see buttons for the shutters for SOLA (the LED light

source for fluorescence), AUX (an auxiliary shutter opened and closed by the software), and DIA (the

halogen light source for bright field). Below these buttons, separated from them by a line, you will see

the Filter Block, which corresponds to the block on the microscope stand itself (see Filter Wheel

Positions lever, above). The choices represent, from L to R: the filter wheel located inside the

microscope, the Dummy position (for color photography or Phase Contrast), the Red filter cube

(excitation 545/30x, emission 620/60x), or the Green filter cube (excitation 480/40X, emission 535/50x).

Filters, Shutters and Switches tab

Below these choices, separated from them by a line, you will see Excitation, and 8 boxes with colored

dots, which represent the different filter cubes. To the right of these last two sections, you will see small

boxes with an arrow pointing towards the upper R hand corner. Clicking on these provides information

about the section in which they are located.

In the dialogue box for the image, you will see tabs along the bottom with labels. During Live imaging,

these will be RGB (merged), Red, Green, and Blue. The merged image represents what you see through

the eyepieces. There will also be information about size (μm/px; RGB 8bit).

Setting up channels for imaging

Go to the area at the bottom of the software screen to the ND Acquisition tab. You can choose among

tabs for T (Time), XY, Z, λ, or Large Image. To set up a multichannel scan, click on the λ tab. Unless you

are using them, ensure that the other tabs are unclicked. Under Set Up, you will see boxes where

channels can be added; press the blue plus sign (+) to add or the red X (X) to remove channels.

New channels will appear under Optical Conf. Use the drop down menu to the right to choose among

objectives, the diastolic shutter (DIA.ILL), or fluorescent filters. Under Name, the software will fill in the

name of the ensuing tab in the Live dialogue box that appears in the main screen. Under Comp. Color,

you can use the drop down menu to choose how each channel appears in the image.

At the bottom of the λ tab, click the Run now button. The software will create an image for each

channel, which will appear in a new dialogue box with separate tabs for each channel as well one for the

merged image.

DS Fi2 (color) camera

On the microscope itself, push the NCB filter into the light path. This filter removes the yellowish cast

that can occur with bright field images. You will be able to see this difference through the eyepieces. The

ND filter does not seem to change the image, although you can try it with your slide/plate/flask/dish to

check whether it helps.

Push the Filter Wheel Positions lever (located on the right hand side of the microscope stand) into the

second position labelled Dummy. This removes any filters from the light path.

In the software:

Go to the DS-Fi2 tab. You will see areas (divided from one another with black lines) labelled Mode,

Resolution, Exposure, and Color, along with buttons and drop down menus to allow you to control how

your image is captured and displayed.

Under Mode, you will see buttons for Normal and Binning. Pressing a button chooses that mode, which

will then be highlighted in green.

Under Resolution, you will see Fast (Focus) and a drop down menu, and Quality (Capture) and another

drop down menu. Below those menus is a box called High Quality Capture that can be checked or

unchecked.

In the dialogue box for the image, you will see tabs along the bottom with labels. During Live imaging,

these will be RGB (merged), Red, Green, and Blue. The merged image represents what you see through

the eyepieces. There will also be information about size (μm/px; RGB 8bit).

Example: to capture an image from a slide of a stained slice of corn stem, I chose the following:

Mode: Normal Fast (Focus): 1280x960 Normal Quality (Capture): 1280x960 Normal – 8bit Exposure: Pressing Auto Exposure button => 184 ms in drop down menu (can be changed) Analog Gain: software sets value AE Compensation: software sets value Color: I had pressed the Auto White button (also appears above live image) to clean up the background Scene Mode >: no action Commands: no action

Miscellaneous

The computer is still linked to the LSUHSC network, but when you log on to use the software, you are logging on to the computer itself, rather than the network. Should you need to log on to the network, close the software and log out of your session. Choose the Log In Another User button

You will need to type in LSUMC-MASTER\username (the one associated with your email) and your regular password. This will take you to a desktop where you can access the network if necessary. You will NOT be able to access your folder with your images, which is on the desktop associated only with the computer itself. Binning averages the grey values in pixels into various sizes of bundles, which makes the signal brighter but reduces resolution. The software allows you to set the binning range for both cameras, with None as the default. The 2x2 selection will produce a good size image for focusing. If you change the binning size, you may need to redo the Auto Exposure. Using 2x2 binning, which averages the information in 4 pixels, increases your S/N ratio by a factor of 4. This is advantageous when you have a weak fluorescent signal. The use of 2x2 binning also decreases image file size and time transfer rates for the camera. Choose No Binning or the lowest value of binning for the best resolution. You can take several snapshots and save them at the end of the session, or save them individually. Keep in mind that crashes can happen. Since NIS Elements is proprietary software, its files will not be read correctly by other imaging software. For instance, Photoshop may open the file as a black screen. To allow software compatibility, you need to save your images as JPEG and/or TIFF files as well. With TIFF files, you need to convert the file to 8 bit format before you save it. If these images are for publication, and as a general tenet of good laboratory practice, it would be wise to always save them in the NIS Elements file format (.ND2). Original formats may be required by publishers and they also contain meta data with image acquisition information. You may not want to convert 16 bit files to 8 bit if you are using your images for quantification or analysis. Tips for Better Imaging Coverslips: It is very important that your coverslip be glass, size 1 ½, 0.17 mm thick. Some of the objectives have correction collars that adjust for differences in thickness, but some do not. The correction collars may help if you are trying to image cells in plates or flasks. However, they may not be in a position ideal for your samples. Plastic: although in general images made through plastic ware are good, it may be worth the extra money to buy optically corrected plastic. Immersion medium objectives: If you are using the 40x, 60x, or 100x oil immersion objectives, please use ONLY the Nikon immersion oil next to the microscope. Residues from different oil types on the lens can cause aberrations in the image. Kimwipes are fine for cleaning the oil off of slides (this can be followed with lens cleaner sprayed onto a Kimwipe, then used to remove the last traces of oil). When cleaning the objective lens, which is very sensitive, use the provided lens paper and draw it gently across the objective, which allows it to soak up and remove the oil without scratching or damaging the lens. For professional quality images, each component of the microscope system should be set to the optimal position for your samples before you begin to collect images. This is especially important for brightfield images that require Kohler illumination. Equipment Specifications for Publication Microscope: Nikon Eclipse TE300 inverted microscope with an epi-fluorescence attachment

Filters: There are Chroma filters in the filter wheel inside the microscope stand, and additional filter cubes mounted separately on the outside of the microscope stand Chroma 8300 filter set with single band exciters

DAPI (360/40x)

FITC (492/18x)

Texas Red (572/23x)

Dual band exciter for FITC and Texas Red Triple band exciter for DAPI, FITC and Texas Red in the Prior filter wheel with a stationary

multiband beamsplitter and emission filter Additional filter cubes

EF-4 FITC HYQ (Ex 480/40x; Em 535/50x)

EF-4 TRITC HYQ (Ex 545/30x; Em 620/60x)

Camera: There are two cameras, one for fluorescence, the other for color

ANDOR Neo/Zyla B&W camera

DS Fi2 color camera Objectives: there are 6 objectives mounted on the turret; the rest are available for check out

Objective Imm Type WD (mm) Use with Where located?

4x/ 0.20 NA Dry Plan Apo WD 20 Microscope turret

10x/ 0.30 NA Dry Plan Fluor WD 16.0 Ph1 DLL Microscope turret

20x/ 0.45 NA Dry Plan Fluor ELWD WD 7.4 DIC L Microscope turret 20x/ 0.50 NA Dry Plan Fluor WD 2.1 Ph1 DLL Microscope turret

40x/ 0.60 NA Dry Plan Fluor ELWD WD 3.7-2.7 DIC M Microscope turret 40x/ 1.30 NA Oil Plan Fluor WD 0.2 DIC H Microscope turret

60x/ 1.40 NA Oil Plan Apo WD 0.21 DIC H Cabinet

100x/ 1.40 NA Oil Plan Apo WD 0.13 DIC H Cabinet

Condenser A Bright field ∞Ph1 Phase contrast

∞L DIC

∞M DIC ∞H DIC

Software: NIS Elements (Windows 7, PC)