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Frequently Asked Questions

FAQs

Q. How do I align my Mercury Bulb for Fluorescence?

Mercury and xenon arc lamps are now widely utilized as illumination sources for a large number of investigations in widefield fluorescence microscopy. Visitors can gain practice aligning and focusing the arc lamp in a Mercury or Xenon Burner with this interactive tutorial, which simulates how the lamp is adjusted in a fluorescence microscope...

For more information [click here]


Q. I would like to buy immersion oil but I don't know which one I need.

Nikon manufacture two types of Immersion Oil for microscopy these being Type A and Type NF. These oils are tested using Nikon objectives and therefore the performance cannot be guaranteed against lenses manufactured by other companies. The refractive indices of other manufactured oils may differ which will impaire the results gained from using these with Nikon objectives. It is also important to note that oils should not be mixed as this will impair the performance.

Type Ais general purpose oil used for imaging applications requiring oil immersion, which can incorporate techniques such as brightfield, darkfield, phase and fluorescence. It is available in three sizes of 8ml, 50ml and 500ml, all supplied with a pipette for dispensing. For a data sheet please [click here].  Type NF is considered to be the superior grade specifically designed for the ever growing need to improve signal to noise ratios in fluorescence microscopy, particularly in low-light applications or those between 340-380nm, typically associated with calcium or deep UV imaging. The improved quality is due to the type of raw materials used in the oil which relates to a subsequent reduction of auto fluorescence caused by minerals in the oil. There is one size available at 50ml, which is supplied with a plastic pipette for dispensing. It is slightly more viscous and has a slight odour. For a data sheet please [click here]. For more detailed information about Oil Immersion please [click here]

To purchase immersion oil please locate you local Nikon distributor from 'Where to buy'


Q. What replacement bulb do I need and can I purchase them anywhere?

The exact bulb you will need will depend on a number of factors which are listed below. The filaments and conductivity of the bulbs are specifically designed for microscopy use. This not only provides a greater evenesss of illumination but also prolongs the life of the bulb. For these reasons Nikon do not recommend non-specific bulbs.

Tungsten (W), Halogen, Mercury (Hg), Xenon (Xe), or Metal Halide

This will determine the type of illumination required but is specific to the type of illuminator the bulb needs to fit into

Voltage and Wattage

This is usually the most significant indicator when presented alongside the microscope model and bulb type. This can usually be found on the original packaging or printed close the contacts.

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Shape or Style

Once you have determined what type of illumination you require, you will need to determine what shape and style. Typical styles of bulbs include oval, rounded and reflective surfaced Example of styles of bulb are attached

Microscope Type

If you are able to determine the microscope model and illumination type this is normally enough to determine what type of bulb is required. Types of illumination include fluorescence, diascopic, teaching head pointer.

To purchase a bulb please locate you local Nikon distributor from “Where to buy


Q. Which objective should I choose?

With the vast array of modern objective lenses currently available, choosing the right type for your application can prove to be a difficult task.

This guide outlines the basic types of objective lenses and identifies criteria for selecting the most suitable lens for your application from basic brightfield microscopy to the more advanced Confocal and live cell imaging studies.

Grades of Objectives

Objective lenses can be split into 3 fundamental groups, Achromats, Fluorites and Apochromats.

Typically, Achromat or Plan Achromat objective lenses are designed to focus blue (about 486nm also called the f line) and red (656nm, c line) in the same plane providing axial chromatic aberration correction. Perfect for basic brightfield microscopy, these are by and large the least expensive objectives to buy.

Fluorites or Plan Fluors are the next grade up and are created from a specially coated glass which allows for higher transmission rates resulting in brighter images. As well as having axial correction for red and blue, fluorite objective lenses are also typically corrected for green (588 nm, also called the d line) along with additional spherical corrections compared to achromats.

The highest grades of available objectives are the Plan Apochromats. In addition to the above corrections, Plan Apochromats are also corrected for the g line (436 nm). Furthermore, Nikon has introduced the Plan Apochromat Violet Corrected series that covers the h line (405nm) as well, totally correcting chromatic aberration for 5 lines, ranging from 405-656nm. Apochromats are typically the most expensive objectives providing the crispest, cleanest images with the least chromatic aberration.

For more information regarding grades of objectives click here

Techniques

Brightfield

Achromats, Fluorites or Apochromats can be used for brightfield imaging. Plan Apochromats produce the best images although Plan Achromats or Plan Fluors can achieve high quality results depending on the application and budget.

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Fluorescence

Fluorescence techniques generally require the use of either Plan Fluor or Plan Apochromat objectives. The perfect objective for fluorescence is very much dependent on each specific application. For example, if the sample has a very weak fluorescence signal, Fluor objectives are more effective.

Advanced Fluorescence and Live Cell Imaging

When activating and imaging fluorescent probes such as CFP, GFP, YFP, Cherry –FP, Tomato FP, PA-GFP, Kaede and PS-CFP, Plan Apochromat Violet Corrected Optics are highly recommended. Axial chromatic aberration has been corrected up to the violet range (405nm), making these objectives highly effective for PA-GFP, as well as other photo-activation and photo-conversion proteins that activate at 405nm. Without violet correction, activation and imaging of multiple probes occurs in slightly different image planes. CFI Plan Apochromat VC optics are also an ideal choice for critical confocal work.

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Plan Apochromat VC lenses not only work well in the violet part of the spectrum but perform well in the IR, enabling greater depth penetration into the specimen. These are also useful for quantum dot deep cell imaging techniques.

Graphs showing the difference in the axial focal position of blue fluochromes when using a standard objective lens and a VC corrected objective lens. Note also the increase in signal to noise.

TIRF

When using Total Internal Reflection Fluorescence, Nikon’s Apo TIRF objectives provide the shallowest evanescent field possible with oil immersion lenses. Apo TIRF objectives provide chromatic correction from 435nm to 1064nm with a N.A of 1.49. In addition they have excellent IR throughput, facilitating the ability to image and trap single molecules in the same focus plane.

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Phase Contrast

A range of phase contrast objectives are available from Nikon to suit different samples.

DL (Dark Low) objectives produce a dark image outline on a light gray background and are suited to specimens with large phase differences e.g. cells. These objectives are a very popular choice for phase contrast.

DDL (Dark Low Low) is a flexible type of objective, capable of being used for multiple techniques including DIC, brightfield, darkfield and fluorescence

ADL (Apodized Dark Low) objectives reduce the unwanted halo effect often associated with phase contrast imaging and allow greater internal detail of specimens such as cells.

DM (Dark Medium) objectives are suited to specimens with a very small phase difference e.g. granules and fine fibres.

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DIC – Differential Interference Contrast

DIC techniques require specialised objectives to complement the DIC prism in use. They are typically Plan Apo or Plan Fluor objectives but contact your Nikon representative to ensure you select a compatible objective to suit your application.

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Polarised light

When using polarised light, strain-free objectives are necessary to eliminate unwanted optical effects caused by physical forces otherwise known as “strain” on the glass elements of the lens.

Other Considerations

Long Working Distance

Working distance can be defined as the distance from the front of the objective lens to the coverglass on the sample when it is in focus. Applications which require the addition of micromanipulators or alternatively use living cells in tissue culture which need to be seen through the walls of thick vessels, benefit from longer working distance objectives.

Multi – Immersion - (Samples with oil, water, glycerine immersion or dry)

A flexible objective lens often useful as a lower magnification companion lens for high resolution immersion lenses especially in Confocal microscopy

Water Dipping – (Samples without a cover slip)

Designed to be submerged directly into the cell culture dish, water dipping lenses typically carry a slightly lower N.A compared to water immersion.

When combined with a long working distance, water dipping objectives are a good choice for micromanipulation applications.

Water Immersion – (Samples with a cover slip)

When live cells or tissues need to be observed deeper in the specimen, water immersion lenses show superior performance with respect to resolution and aberration correction. Designed to be used with a cover slip, the optical path is kept symmetrical as light passes from cells (watery) to glass (cover slip) to water again (immersion medium) and then to glass ( objective lens) again.

Correction Collars

Correction collars enable the user to adjust the optics to account for certain parameters such as cover glass thickness, immersion medium, refractive index, temperature and depth penetration into tissues, thereby achieving optimum performance.

To discuss your requirements further please locate your local Nikon distributor from “Where to Buy”


Q. What camera mount (c-mount) do I require to connect my camera to my microscope?

There are literally hundreds of potential combinations with many different camera and microscope types. The most fundamental information you will need when deciding which c-mount adapter you need are as follows:

What make and model is your microscope?

This information is important as there will be different focal positions (point at which image data will be collected on the cameras chip) for different manufacturers microscopes. Modern microscopes will tend to have the model type on the microscope body, sometimes near the serial number. As an alternative the microscope manufacturer should be able to identify the microscope by its description or by a photograph.

What make and model is the camera you are trying to attach?

This is an important piece of information as it usually determines the type of thread or fitting the c-mount adapter should have.

What port will you be using?

It is not always possible to connect a camera via a dedicated camera port (sometimes referred to as trinocular tube or beamsplitter). It is important that you identify which port will be used to accept the c-mount.

Is a magnification necessary?

c-mount

The camera chip (CCD) will be of a certain dimension and will require the appropriate magnification within the c-mount adapter which is sometimes referred to as a relay lens. A good rule of thumb is that whichever size the CCD is as a decimal is what it requires in magnification to fill the viewfield. For example a 2/3” CCD (0.67”) requires a 0.7x c-mount. The CCD size should always be made available by the manufacturer.

It is always remembering that a lower magnification will give a wider field of view and generally brighter images for shorter exposures. For an example of the effect of different size images click here

To discuss your requirements please locate you local Nikon distributor from "Where to buy"


Q. Which filter blocks should I use for fluorescence imaging?

This will depend on the fluorescent probes that you intend to use in your imaging application.

The filter block houses an excitation filter, dichromatic beamsplitter (mirror) and a barrier (or emission) filter, which need to be matched to the excitation and emission wavelengths of your probe(s). You will need to know the excitation and emission wavelengths of your probes before selecting the appropriate filter / dichroic combinations. This information is available from your probe supplier.

For a quick and simple-to-use Nikon filter block selector matched to specific fluorescent probes [click here]

For further information on fluorescence filter combinations visit [click here]

To discuss your requirements please locate you local Nikon distributor from "Where to buy"


Q. Which digital camera should I select for my application?

As digital technology has improved, so have the range of cameras and the complexity of choice. A few years ago, particularly for consumer cameras, it was popular to consider the number of pixels to be a guide as to which camera was best. The current level of understanding is much deeper now and this raises a number of questions that need to be answered before an appropriate camera can be purchased. To help you select the best camera for your requirements we have listed a series of further questions which should help you discover what camera is right for you and your application.

Consumer vs Microscopy Camera

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The biggest factor which often drives this question is price and convenience. Quite often a consumer camera is purchased for microscopy work because of the perceived cost saving, the larger number of pixels and the ease at which it can be used off the microscope. These are great attributes in isolation but will not provide the best images possible from your microscopy system.

In pursuit of increasing the number of pixels, consumer cameras tend to utilize cheaper CCD’s which are likely to have a higher number of defective pixels. These defective pixels manifest themselves as Hot Pixels, Stuck Pixels or Dead Pixels.

Dedicated microscopy cameras tend to have larger pixel size which reduces the number across the size of CCD. However larger pixels provide a greater signal to noise ratio and ultimately image quality.

The glass and lenses used in consumer cameras are not designed to integrate with microscopy glass. Dedicated cameras do not have built in glass and are therefore are more compatible by virtue of a dedicated c-mount.

Consumer cameras tend to have preset functions that cannot be turned off i.e. sharpening and contrast enhancement

Dedicated cameras are more adapt to more advanced microscope imaging techniques through dedicated software and techniques such as telemicroscopy.

Monochrome vs Color

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This question is usually raised when sensitivity or speed are of primary importance. Before determining what type of camera you need you should identify what type of results you require.

Single CCD or monochrome cameras have no color assigned to their separate pixels. Monochrome cameras tend to have larger pixels which invariably mean fewer pixels on the CCD. This increases their sensitivity and speed at which they can gather imaging data. For this reason they are more suitable for fluorescence imaging i.e. documenting cellular interactions.

Color cameras employ color filters which overlay the pixel and are Red, Green or Blue. This is particularly useful for applications where color fidelity is key i.e. stained tissue samples or polarization microscopy.

Pixel Size vs Pixel Number

CCD’s are generally manufactured in three sizes 1/3”, 1/2” and 2/3”. As the size of the CCD is fixed the key is to utilize the space with the appropriate pixels.

The greater the number of pixels squeezed on the CCD, the more detailed information can be gathered. This is more relevant for low magnifications or macro work. This does mean that there is a greater incidence of pixel failure with the possibility higher noise degrading the image.

The larger the pixel size, the less number are able to fit onto a CCD and a greater sensitivity is achieved. This is more relevant to higher magnifications where less area is recorded or imaging work requiring a high signal to noise ratio.

Resolution vs Speed

This is the biggest trade off in microscopy imaging. As the need to gather greater information or resolution increases, invariably the speed at which the signal data is collected and assimilated slows down.

Fast imaging systems will use a lower area or number of pixels, thereby increasing their frame rate or speed of data acquisition

The faster the acquisition the less time there is to gather detailed information and therefore resolution or image data volume is reduced.

Cooled Options

To further enhance the signal to noise that can be achieved it is possible to purchase camera options which have a cooled CCD.

The CCD is either cooled to a specific temperature or a temperature set at a certain level below ambient temperature.

Cooling of the chip enables a reduction in interference caused by temperature fluctuations and noise.

Cooled CCD’s are found on Monochrome cameras and some color cameras.

To discuss your requirements please locate you local Nikon distributor from "Where to buy"


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