Apr 27, 2026
Nikon Advanced Microscopy - Confocal Reflection Label-Free Imaging
- 1Nikon
- Nikon Healthcare UK, Branch of Nikon Europe
Protocol Citation: Akihiro Suto 2026. Nikon Advanced Microscopy - Confocal Reflection Label-Free Imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3mn77l25/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 31, 2026
Last Modified: April 27, 2026
Protocol Integer ID: 314220
Keywords: confocal reflection microscopy, free imaging confocal reflection microscopy, nikon advanced microscopy, depth explanation of confocal reflection microscopy, allowing reflection microscopy, reflection microscopy, nikon ax confocal tunable detector, fluorescence imaging, reflection imaging, confocal reflection label, confocal tunable detector, fluorescence, opaque substrate, like imaging, nikon axe, laser light, autofluorescence, substrate, stained sample, cell, label free brightfield
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Abstract
Confocal reflection microscopy captures laser light reflected from samples, and allows structures to be visualized without staining or autofluorescence. It can be used to study, for example, biomaterials, cell-growth surfaces, beads within cells, or structures such as blood vessels. Confocal reflection microscopy can be combined with fluorescence microscopy to study interactions between stained samples and the substrate onto which they are mounted. Additionally, confocal reflection microscopy allows the study of samples mounted on opaque substrates, allowing label free brightfield-like imaging. Using the Nikon AX confocal tunable detectors, it is easy to set up reflection imaging, and does not require additional hardware such as dedicated filter cubes, allowing reflection microscopy to be paired with fluorescence imaging. A more in depth explanation of confocal reflection microscopy can be found here: Confocal Reflection Microscopy | Nikon’s MicroscopyU
Troubleshooting
Problem
Reflected Signal is weak
Solution
The reflected signal strongly depends on the reflectiveness of the surface as well as on
the qualities of the primary dichroic mirror. The blocking of the laser line by the AX dichroic mirrors is very good, such that leakage of the laser line with the use of these may be only possible for highly
reflective surfaces (e.g. Metal.) When using a weakly reflective sample, it can be better to use the 20/80 beamsplitter dichroic, which allows 20% of the laser light to reach the sample while letting 80% of emissions through.
This may cause an image shift between the reflected image and the fluorescence images, in which case the "Channel Alignment and Registration" tool within NIS elements may be used.
Alternatively, the fluorescence channels can be imaged with the 80/20 beamsplitter, at the expense of fluorescence intensity.
See Troubleshooting section at the end of the protocol for diagrams.
Problem
There's a bright spot in the centre of my image
Solution
This can occur with reflected imaging due to backreflections not caused by the sample. It is recommended to move the confocal scan area to a corner of the sample.
See Troubleshooting section at the end of the protocol for diagrams.
Safety warnings
Please note, the reflection signal in confocal imaging can be very bright and usually the laser power needs to be very low for this channel. Be careful not to overexpose the detector by starting with a low gain and gradually increasing the gain. The software will automatically set the laserpower and gain to a low value when taking a reflection image.
While there are safeguards in place to protect the photomultiplier tubes from being overexposed and damaged, Nikon does not take responsibility for accidental damage of the PMT due to careless usage during reflection microscopy. Use this protocol with caution!
Setting up Reflected Imaging
Place sample onto microscope stage.
Add an experiment on the AX Confocal
Select Multi-channel Experiment
Select Based on Dyes
Add in fluorophores present in sample, then press finish.
Enter Expert Mode in experiment configuration to add a reflected channel
Add an extra channel to the experiment, and select reflected acquisition toggle.
Here, the channel can be renamed and a false colour applied, for example as below.
Configure the reflected channel.
N.B.Due to the architecture of the detectors, reflected imaging with the 405nm lasers is not possible simultaneously with DAPI detection on the first detector. If this is required, then a two-pass experiment must be configured
The immediate result after pressing "OK" in the previous step - The reflected channel needs to be reassigned to a tunable detector, GaAsPs in position 2 or 3 in the list.
Cut the connection (by clicking on the line) from the connected channel to the 4th detector, and re-add this connection to GaAsP detectors 2 or 3.
Drag and drop from the red box onto the GaAsP detector, as below:
Before reconnection
After reconnection
Set the reflected channel to be imaged on a separate pass (frame pass)
Extend the variable filter range to cover the laser excitation for the reflected channel
Save the experiment
Slowly increase the gain and laser power until you can visualise the reflected channel.
Please note, the reflection signal in confocal imaging can be very bright and usually the laser power needs to be very low for this channel. Be careful not to overexpose the detector by starting with a low gain and gradually increasing the gain. The software will automatically set the laserpower and gain to a low value when taking a reflection image.
While there are safeguards in place to protect the photomultiplier tubes from being overexposed and damaged, Nikon does not take responsibility for accidental damage of the PMT due to careless usage during reflection microscopy.
Expected result
Note: These results are examples of a fixed Bovine Pulmonary Arterial Epithelial slide, imaged with a 20x Air objective on the Nikon AX confocal microscope, with confocal zoom and cropping to show fine details within a cell. The 20/80 beamsplitter was used for the confocal reflection channel, and the standard dichroic was used for the fluorescence channels, with image registration in NIS elements post-acquisition.
Confocal Reflection image only. A 640nm laser line was used for reflection - no fluorescent signal is observed with the 640nm laser in this sample.
Confocal Reflection image merged with Fluorescence images.
MitoTracker‱ is shown in red, Alexa Fluor‱ 488 Phalloidin is shown in green, and DAPI is shown in blue.
Troubleshooting - Weak Reflected Image
Open up experiment setup in expert mode and click on the first dichroic mirror.
Select 20/80 beamsplitter dichroic
Troubleshooting - Bright spot in centre of field of view
Move the scan area away from the centre of the field of view.
Troubleshooting: Image Registration in NIS Elements
Please see user manual here: NIS-Elements User Manual
Image registration requires the image to have a structure that is present in both channels for registration.
It is only required if switching primary dichroic mirrors between the reflected channel and fluorescence channels.
The specific section containing image registration can be found here: Image Registration | NIS-Elements