Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

We first presented iSCAT in 2004 in the context of detection and spectroscopy of gold nanoparticles. In the 10 years that followed we developed this technique further for detection and tracking of biological nanoparticles like viruses and small proteins. The essence of the technique is that any material object, no matter how small, has a finite extinction cross section.

The main advantage of this technique is the label-free detection. This means that if we're sensitive enough, we can detect just about anything, like proteins or exosomes secreted from single cells. The issue that one has to be careful about is how to treat the scattering background.

A great advantage of an iSCAT microscope is that it can be completely home built and it can be added to an existing commercial microscope. This means that it can be easily combined with other optical techniques, such as fluorescence, and this is one of the reasons that many groups are also now employing iSCAT and related techniques. Here we utilize LUZ cells as a model system to show the detection of individual and secretory proteins.

However, this method can be also applied to probe almost any biological process at the molecular level. To achieve a stable microscope, begin with a damped optical table and a rigid massive block for the sample stage. Build a microscope sample stage that incorporates a high numerical aperture objective and a translation unit that allows for lateral sample translation as well as change of focus position for the objective.

Use a 45 degree vertical coupling mirror and a 50 centimeter focal length singlet lens to focus the light of a diode laser at wavelength 445 nanometers onto the back focal plane of the objective. This wide field lens creates a collimated beam at the objective's forward focus, which will become the iSCAT illumination source. Apply a droplet of immersion oil to the objective and place a glass coverslip in the sample plane of the microscope stage.

This will result in a beam that reflects back down through the imaging objective. To set up the imaging path, introduce an anti-reflection coated beamsplitter at a 45 degree angle relative to the incident beam and approximately 10 centimeters after the wide field lens. Make sure that the anti-reflection coating points towards the laser source.

Pay attention as a thick beamsplitter will introduce significant beam displacement so that the laser might not enter the objective straight anymore. If necessary, realign the laser beam path before the beamsplitter to ensure the correct propagation through the objective. To ensure that the sample plane and camera are parfocal, start by placing a concave lens with a negative focal length of 45 centimeters at a position five centimeters after the wide field lens in the incident beam path.

This will result in a collimated beam entering the back aperture of the objective. With the screen placed in the reflective arm of the interferometer, move the objective in the vertical direction to find the coarse focal position. The objective is in focus when the beam hitting the screen is collimated.

Remove both the negative focal length lens and the screen when coarse focusing is completed. Add a second 50 centimeter focal length singlet lens to focus the scattered light and to collimate the reflected light. Ensure that the lens is placed 50 centimeters from the back focal plane of the objective so that the transmitted laser beam is collimated again.

To complete the iSCAT setup assembly, place the CMOS camera 50 centimeters away from the 50 centimeter focal length lens, and position the beam directly onto the middle of the chip. To set up additional imaging channels, couple the output of an LED light source into a long working distance objective. Install mechanical components above the sample chamber that allow for focusing and lateral positioning of the LED output onto the sample.

Move the upper objective laterally so that the upper wide field objective and the lower iSCAT objective are colinear. This is determined by placing a screen under the lower objective and maximizing the intensity of transmitted LED light on the screen. Now, place a 550 nanometer shortpass dichroic mirror to split the transmitted LED light from the iSCAT laser path.

Split this beam into two channels with an eight percent reflective, 92 percent transmissive, beamsplitter. The 92 percent path is the fluorescence channel, and the eight percent path is used for brightfield imaging. Image the brightfield channel onto a CMOS camera using a five centimeter focal length achromatic doublet lens.

Image the fluorescence channel onto a separate CMOS camera using a five centimeter focal length achromatic doublet lens. Also, use a 600 nanometer longpass filter to block the excitation light. To set up the computer and software, connect all cameras to a computer.

On the fully assembled setup, observe the iSCAT image on the CMOS camera and ensure that it is in focus by finding a residual dust or dirt particle on the glass coverslip. Verify that the particle's image is a circularly symmetric point spread function. The point spread function will not have a circular shape if the laser beam enters the microscope objective at a slight angle.

This can be corrected by slight adjustment of the 45 degree mirror to ensure straight coupling into the objective. Compare the camera images of the brightfield and the fluorescence channels. Ensure that both are in focus, and display the same area by imaging a fluorescent bead or cell sample.

Verify that the position of the iSCAT laser is approximately in the center of the image, and take note of its position for later reference. To change the position and the field of view of the brightfield and the fluorescence channel, move the cameras on the table with respect to the focusing lenses. Prepare for the experiment as detailed in the text protocol.

This includes preparation of the cells and microscopy medium, as well as the microscope sample cuvette. Ensure that the laser beam is blocked to prevent the cells from being directly exposed to the iSCAT laser light. Inject approximately three microliters of the prepared cell sample slightly off center into the sample cuvette.

Gently touch the pipette tip to the coverslip and slowly inject the cell solution. Allow the cells to settle on the coverslip. Check the number of cells close to the iSCAT laser.

If the number of cells is too low, repeat this step until a sufficient number is available. If the coverage of cells is too dense, use an injection of approximately 20 microliters of additional microscopy medium to disperse the cells across the coverslip. Using the piezoelectric translation stage, move the sample laterally to position a cell close to the iSCAT field of view.

Ensure that the cell does not enter the iSCAT field of view as direct exposure to the 445 nanometer laser light might be harmful for the cell. Unblock the iSCAT laser beam and ensure that the coverslip surface is still in focus. Enclose the isolation table to minimize drift and acoustical coupling from the ambient surroundings.

Start the measurement by acquiring images from the iSCAT, brightfield, and fluorescence cameras. Periodically check the viability of the cell and the focus of the system. Here, self-written microscope software is used to display the camera images.

Here, differential imaging is performed in real time by subtraction of consecutive frames to make protein bindings visible. This results in a filtered image that is visible on the screen together with the raw camera image. Representative results of a cellular secretion experiment carried out with iSCAT are shown here.

The video shows secretions of a LAZ cell over the course of two minutes. Differential iSCAT images on the left visualize the absorption of single proteins to the coverglass. The brightfield images and fluorescence channel on the right are used to monitor cell viability.

This histogram shows the detected proteins and their contrast range within that two minute time period. The data was collected by analyzing individual binding events in each frame of the iSCAT video data using a custom peak seeking algorithm. ISCAT microscopy is not only a powerful tool in biosensing, but also in microscopy, because it allows for label-free detection of nano-objects in real time.

In particular, it can be applied to a variety of processes such as diffusion and transport of proteins. Due to its exquisite sensitivity to light scattering, iSCAT can detect any protein or entity in the field of view. Of course, this also means that the technique lacks the specificity that fluorescence brings along, but to get around this issue, one can apply additional methods like surface functionalization to detect specific proteins of interest.

Don't forget that working with lasers can be dangerous, and appropriate eye protection should always be worn when assembling and adjusting the microscope. Real time detection of secretomes is very exciting and a major leap in medical diagnostics, which currently requires much longer time and is very far from single protein sensitivity. There is still plenty of room for improving the performance of the method and extending its applications.

So we hope that this video helps other groups join this exciting effort.