Advanced Nanoparticle Tracking: Enhanced frame rates and resolution with NanoSight Pro

Introduction

Nanoparticle Tracking Analysis (NTA) is a powerful technique used to characterize nanoparticles in suspension by visualizing and analyzing their Brownian motion. This method leverages advanced optical microscopy and sophisticated software to track the movement of individual particles, providing detailed information on particle size distribution, concentration, and dynamics.

A critical aspect of NTA is the performance of the camera system, particularly its frame rate and resolution. The frame rate, measured in frames per second (fps), determines how frequently images are captured, directly influencing the accuracy of particle tracking and the temporal resolution of the analysis. Camera resolution, on the other hand, defines the level of detail captured in each frame.

Balancing frame rate and resolution is essential for optimizing NTA performance, ensuring reliable and reproducible results across a wide range of nanoparticle sizes and concentrations.

This technical note explores the impact of variable frame rate and camera resolution on NTA, providing insights into best practices for selecting and configuring camera and recording conditions to achieve optimal analysis outcomes with NanoSight Pro.

NTA principles

Nanoparticle Tracking Analysis (NTA) with the NanoSight product line utilizes the properties of light scattering and Brownian motion to obtain high-resolution particle size distribution in liquid suspension. Particles are contained by a top plate over a laser module and illuminated by a laser beam causing them to scatter light. This light is then observed with a high-sensitivity camera and a 20x objective lens to look at the particles’ mean-squared-displacement (MSD) on a frame-by-frame basis. This, in turn, is converted to the hydrodynamic diameter through the Stokes-Einstein equation - see below - accounting for the temperature (T) and viscosity (η). 

The process of analysis entails 

1. recording a video of your particles Brownian motion
2. tracking each particle’s movement on a frame-by-frame basis 
3. processing the data to plot concentration vs size as shown below (Figure 1):

Image optimization 

Camera setup

The quality of the captured videos is fundamental to obtaining robust, reliable data. As such, understanding the optimal conditions for image and camera setup is essential for generating accurate tracking results. Ideally, the setup should produce well-defined, sharp dots against a clean, dark background. 

NanoSight Pro facilitates this by offering both automatic and manual camera setup options, allowing users to adjust two key parameters: exposure time and contrast gain. 

• Exposure time determines the length of time the camera’s electronic shutter is open, thereby controlling the amount of light captured from the particles. This setting directly influences the frame rate. 
• Contrast gain enhances the camera sensitivity and should be increased to allow the smallest particles present in the sample to be visible and capable of being tracked and analyzed. 

The relationship between exposure time, contrast gain, and frame rate represents a significant improvement over legacy systems that operated with a fixed camera rate of 25 frames-per-second (discussed below) and adjusting the camera levels.

Recording time: from seconds to frames

For years, the NanoSight product line operated with a fixed camera rate of 25 frames per second. Previously, the general recommendation was to adjust recording duration, in seconds, to capture the optimum number of data points for either a more polydisperse sample or a dilute sample. For example, a 60+ second recording was typically a utilized SOP for the  majority of polydisperse or dilute samples.  This approach allowed for the collection of statistically relevant data points per bin size across the size distribution. Conversely, fewer seconds of recording were sufficient for more monodisperse samples. 

The new NanoSight Pro and its NS Xplorer software (Figure 2) have transitioned from focusing on the total duration of each video to utilizing a variable frame rate. NTA tracks the positions of each particle over several frames to better statistically understand their Brownian motion behavior. The NS Xplorer can now record videos in less time compared to the NS300 and older models by adjusting the frame rate while maintaining the same statistical significance. The variable frame rate ranges from 25 frames per second (fps) to 65 fps, unlike the fixed 25 fps of previous NanoSight models. The frame rate is determined by the exposure time setting. As shown in the figure below, shorter exposure times result in higher frame rates.

Example data includes the following:

• Maximum exposure time of 40 ms = 25 fps
• Exposure time of 23.7 ms = 42 fps
• Exposure time of 15 ms and below = >65 fps

The relationship between exposure time and frame rate further translates to the total recording time for the video. This is further shown below where the time it takes to capture a total of 1500 frames varies with camera exposure time: 

To review, let’s look at two different scenarios:

1. If you have a lower concentration or dim particles, then you would expect:
   Long exposure time = Low frame rate = Longer video duration
   
   
2. If you have a higher concentration or bright particles, then you would expect:
   Short exposure time = High frame rate = Short video duration. 

It is worth noting that 750 frames per recording is sufficient for most sample types to generate a statistically robust analysis, so the actual duration is between 10-30 seconds per recording.

If we extend this theory and method to auto settings you would find that the default auto setting for scatter mode is 750 frames where:
@25 fps = 30 s
@65 fps = 11 s

For a fluorescence mode measurement, a higher frame rate allows for enough signal from photobleaching samples with only 150 frames:
@25 fps = 6 s
@65 fps = 2.3 s

The default values of number of frames per recording can be always adjusted by user to make sure that enough particle tracks are generated, thereby obtaining statistically significant data.

Camera – new capabilities

Obtaining a perfect image of white dots on a perfectly dark background is just one component of successful tracking. The next step involves the software's ability to distinguish particle intensity from the background. This can be challenging for the smallest and dimmest particles, which often have brightness levels similar to their surroundings, making it difficult for the algorithms to detect and track them. This is where utilizing the full potential of the camera resolution becomes extremely beneficial.

Unlike the NanoSight Pro, older NanoSight systems used 8-bit greyscale values affording 256 grey-levels (28 = 256) between black and white as seen below in Figure 5. The new NanoSight Pro uses the full capability of the camera’s native 12-bit resolution. This affords 4096 grey-levels (212 = 4096) between black and white (Figure 5).

Think of a 12-bit camera resolution like having a box of 4,096 different colored crayons. Each crayon represents a different shade of color that the camera can capture. The more crayons you have, the more detailed and nuanced your picture can be. In the same way, a 12-bit camera can capture 4,096 different levels of brightness, allowing it to distinguish very fine differences in light and dark areas. This high level of detail is especially useful for detecting and analyzing small, dim particles in Nanoparticle Tracking Analysis.

This offers 16 times the greyscale resolution of the NS300. So how does resolution affect the image processing? This means that software can detect finer fluctuations in the gray level and thus allowing the system to detect dimmer particles and particles partially obscured by noise. This further strengthens the use of the NanoSight Pro’s machine learning model – you can read about this in the blog listed below.

Summary

By harnessing the full potential of the camera, including the implementation of high frame rates and 12-bit resolution, NanoSight Pro significantly reduces capture time without compromising data quality or the statistical significance of particle tracking. The high frame rate ensures that even fast-moving particles are accurately tracked, while the 12-bit resolution provides a detailed and nuanced capture of particle intensity, enabling the detection of even the smallest and dimmest particles. This combination allows for efficient and precise nanoparticle analysis, making the NanoSight Pro a powerful tool for researchers seeking reliable and reproducible results in less time.

Resources:

• NanoSight Pro
• Nanoparticle Tracking Analysis
• Blog: Nanoparticle Tracking Analysis with Confidence! – The use of Machine Learning with the NanoSight Pro