Open-Hardware Without Compromise
Single molecule microscopy can reveal both sub diffraction limited structures and molecular dynamics directly in living cells but, unsurprisingly, requires advanced microscopes to do so. Commercial microscopes are available that provide these capabilities but are expensive and inflexible. In comparison, home built systems are almost infinitely modifiable but require a significant level of expertise and time to create.
Open-source microscopes have become increasingly popular as a way of reducing this initial setup barrier while retaining the flexibility of home built systems. However, open-source systems have often focused more on this aim of accessibility at the expense of a full feature set.
In its base form, the LifeHack microscope is fully capable of SMLM and live cell imaging. Unlike commercial microscopes however, it is intended to be equally useful as a development platform from which to build specialised systems to solve cutting edge problems. In comparison to existing open source microscopes, the LifeHack is particularly advantageous for live cell imaging. This is due to its stable, super-resolution microscopy compatible, temperature control plus robust hardware autofocus/live drift correction in all 3 dimensions over a large depth range.
LifeHack has been constructed from commercially available and 3D printed parts to simplify the build process and comprehensive parts lists, CAD files, and build/setup instructions are available to allow straightforward construction of the microscope. The complete parts list can be purchased for approximately £150,000 (as of 2020).
Features
| Requirement | Feature |
|---|---|
| Stable Temperature | Custom Incubation Box |
| High Signal to Noise Ratio | Ring TIRF / HILO |
| 3D Tracking | Variable Strength Astigmatism |
| Multi-Channel SMLM | 4 Lasers + AOTF Switching |
| Focus Lock | Extended reflection based + IR Brightfield based Autofocus |
| Adaptable | Modular and Open Source Designs |
Design Philosophy
To achieve the goal of creating a system that is adaptable and future proof we have designed the microscope around 3 main modules and 3 sub-modules. These modules may be modified individually or used in conjunction with other systems. The system is almost exclusively made from either commercially available components or 3D printed parts with ease of reproducibility in mind.
The designs for the microscope have been modelled using Autodesk's Fusion 360 software. These designs allow for users to adapt the system to suit their need and function as easy to follow plans during the build process. The entire set of CAD files for the microscope are readily available to view online and download from each module's page.
👁 View CAD Design Online 👁Live Cell Capable
An incubator design enclosing the main body of the microscope is included as part of the system with a wide array of access hatches for both sample manipulation and microscope alignment. This not only fulfils the essential requirement of keeping living samples at an optimal and consistent temperature to grow but also provides improved environmental stability for the microscope body thereby drastically reducing drift during imaging.
Extended Focus Lock
The Microscope is capable of providing drift correction using a reflection based autofocus system. As cells are three dimensional and imaging only at the coverslip is not sufficient, this system is augmented by a translatable tube lens that extends the lock range to approximately 10μm. This allows for investigation of internal cellular structures.
The LifeHack system additionally includes an infra-red camera and LED to enable brightfield imaging concurrently with fluorescence imaging. This means that image or bead based drift correction can be implemented without affecting data collection. This camera based system also provides autofocus lock even deeper into samples (50μm) as demonstrated in "FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in bacterial cell division", Whitley et al, bioRxiv 2020. This has now been extended to provide live drift correction across all three dimensions and to work in conjuntion with the reflection based system.
Ring TIRF for Excellent Signal to Noise
TIRF and HILO illumination are provided by the incorporated pair of Galvo-scanning mirrors. These mirrors are capable of oscillating rapidly which allows for the beam to be scanned in circles at 100s of Hz. This enables the use of ring TIRF whereby through the course of an image the beam illuminates the coverslip from all angles. Shadowing or artifacts caused by imperfections in the coverslip or inhomogeneities in the sample are thereby reduced.
Astigmatic 3D
3D localisation is provided by a cylindrical lens positioned in a 3D printed box immediately before the camera. This contains a rail mounting system to allow for axial adjustment of the lens position. Sliding the lens towards and away from the camera will respectively reduce and increase the severity of the astigmatism at the camera. A stronger astigmatism enables more accurate localisation in z as the ellipticity of the PSF will change more rapidly as the source position does.
High Power Multi Colour Imaging
The system is capable, in its base form, of simultaneous 2 channel and sequential 4 channel microscopy. This is achieved by its 4 lasers (405nm, 488nm, 561nm, 642nm) in the excitation module. The Lasers range in maximum power from 100mW to 1W and follow an open beam path from source to sample for greater efficiency.
Structure
The LifeHack System is organised into 3 main modules:
- Excitation module: Contains the the lasers and AOTF / Shutter for exposure control.
- Beam Expansion module: Raises the excitation beam to the correct height for the back port of the microscope and uses a negative/positive lens pair to expand the beam diameter.
- Main Body: Includes the necessary structure for the optical paths, sample stage and camera for imaging. This includes components to allow for ring TIRF and IR based autofocus systems.
The microscope also includes three sub-modules that are associated with the Main Body of the system but can be incorporated separately into another system to add functionality:
- Incubation Box: A custom acrylic box enclosing the main body. Includes large hatches for easy sample and hardware manipulation.
- Focus Shifter: Normally encompassed by the main body but effective with any system. Extends the working range of reflection based autofocus systems using a translatable tube lens.
- ImLock: Normally encompassed by the main body but effective with any system. Offers deep IR-brightfield image based autofocus and lateral drift correction along-side fluorescence data collection.
Change Log
- 24th February 2021: Initial release of all modules
- 1st April 2021: Reduced diameter of 100mm beam expansion lens from 2" to 1"
- 10th May 2021: Changed suggested operating wavelengths of ImLock(940nm → 850nm) and Focus Shifter(850nm → 940nm)
- 4th April 2023 Major update to ImLock
- 20th November 2023 ImLock v2.0.0 Release