Lasers
What is the estimated lifetime of the laser?
The laser is based on a fiber laser architecture with a free-space optical section at the output. The laser is pumped with industry-rated pump modules and is thoroughly tested before shipment. The standard guarantee is 24 months on the microscope and laser, which can be extended to a total of 5 years. As the entire laser engine can be replaced in the field, downtime of the microscope in case of failure is minimized.
What repetition rate for the lasers do you offer?
100 MHz. Other repetition rates on request, like 80 MHz.
What output power do you measure directly at the laser output?
Typically, > 500mW average for the tunable and fixed line fs laser.
What output power do you measure after the objective on the sample?
Typically, >150mW, depending on the specified power in the purchase order and depending on the objective.
Can your lasers be used for other microscopy techniques than two-photon?
Yes! Our Lasers can also be used for CARS, SRS, FLIM, optogenetics, (please find an application note about CARS/SRS imaging in our download folder)
Do you offer lasers for three-photon (3P) microscopy?
No, but our MPC 3P-ready microscope body allows to integrate any external 3P laser.
Do your lasers have an option to chirp or for active pulse compression?
A motorized dispersion compensation module is built in.
How is the laser connected to the 360-Frontend?
The laser is connected to the 360-Frontend with a polarization maintaining single mode fiber.
What is the typical pulse duration?
Typically, <140 fs at the specimen, check quote and purchase order specifications.
Can I use my own laser?
Yes! If you already have your own laser or if you want to use the microscope for 3P imaging, we have our MPC series, which allows you to integrate your own laser. The MPC has a coupling port for external lasers and is optimized for wavelengths from 680nm up to 1700nm (THG and 3P imaging).
Scanners & Detectors
What type of scanners do you offer?
We offer linear galvo-galvo scanners in the basic version that can be upgraded with an 8kHz resonant scanner, which allows in combination Resonant-Galvo-Galvo scanning. Resonant-galvo-galvo means 3 consecutive scanners in the system and allows rapid multi-region scanning. A detailed explanation can be found here:
What is the frame rate with the galvo-galvo and the resonant scanner?
The frame rate always depends on the imaging-parameters like pixel count and dwell-time. For an image with 512×512 pixel and a dwell-time of 3.2ms, the frame rate for high quality imaging the linear scanner is approx. 1 Hz and for the resonant up to 30Hz fps.
Line scan, single point?
ScanImage also allows to do line scans or keep the laser on a single point.
What kind of detectors do you use for the two-photon detection?
For the 2- & 3-Photon and SHG/THG-detection we use highly sensitive, specially selected low-dark-count rate GaAsP Photomultiplier tubes (PMTs) from Hamamatsu.
Why do you use PMTs instead of APDs?
PMTs offer a much larger detector size and thus enable high contrast, low-light, deep imaging.
Do you use the PMTs in an integrated or gated mode?
Typically, PMTs are used in integrated mode, but gated mode can be implemented.
How many detection channels do you offer?
We offer up to 4 PMTs in non-descanned mode.
Objective & FOV
What field of view (FOV) do you have for multiphoton fluorescence?
For the multiphoton the FOV can be easily changed via changing the scan angle of the galvo mirrors. The maximal intended FOV here is 750 μm for the 20x objective but can be derived up to 900 μm. However, within this FOV, shadowing effects at the edges can occur. We strongly recommend using the intended FOV. FOV of the 16x objective is about 900 m.
What objectives can be used in your microscope?
All infinity corrected objectives (Nikon, Olympus, Thorlabs) can be used for the widefield path – others (Zeiss, Leica) will change the FOV. For the multiphoton imaging, the objective should be transmissive in the near infrared (up to 1000 nm for two-photon and up to 1700 nm for three-photon). Moreover, the resolution for non-linear microscopy techniques like two-photon depends on the NA and not the magnification! So please use objectives with a high NA!The thread in the objective turret is M32 x 0.75 for all 3 positions.
What objectives can be used for three photon microscopy?
Good transmission up to 1700 nm is a key element. Nikon and Olympus objectives with long working distances are suitable for that.The thread in the objective turret is M32 x 0.75 for all 3 positions.
Do you have a variable beam-expander for different objectives?
No, the beam is aligned to fill the 5mm galvo mirrors, which translates into a 22mm beam diameter at the objective back aperture.
For what aperture size is the beam size optimized?
About 22mm diameter. Any objective smaller than this will truncate the beam, resulting in a power loss. However, our design will always make us of the full NA of modern, state-of- the-art objectives with large working distances, high NAs (typically >1) and large back aperture sizes.
What happens when I use an objective with a bigger or smaller aperture?
You can use both objectives with a bigger and smaller aperture which will lead to an over- or underfilling of the back focal plane. With overfilling you might lose a little laser-power due to clipping, by underfilling you might lose resolution, but you might gain a higher penetration depth.
Is SHG also fluorescence?
No. SHG works without fluorescence markers, that ́s because it’s one of the “label-free” imaging techniques.
Is the FOV the same for the widefield and the multiphoton?
The FOV for the multiphoton scanning depends on the scan angle of the galvos (which is variable). You can calibrate both FOVs to be the same size, but they will always be shifted a little bit to each other.
Imaging: dwell time, rep rate and fps?
The dwell time defines how long the Galvo position will stay at each pixel. This time multiplied by the resolution gives (round about because of flyback times) the time per image, the reciprocal value of the frames per second (fps). With the given dwell time and laser rep rate, the number of pulses per pixel can be calculated.
Note: Galvo-Galvo scanning:
For Galvo-Galvo scanning a dwell time of 3.2 μs is the optimal experience value so far. The dwell time is independent from the resolution. When doubling the resolution, the fps is divided by four.
Note: Resonant-Galvo scanning:
The resonant scanner has a fixed frequency of 8 kHz. Therefore, the dwell time is only dependent on the resolution. When increasing the resolution, the dwell time will decrease so the scanner frequency will stay the same. When doubling the resolution, the fps is halved.
Multiphoton detection filters
The optical density (OD) describes the remaining transmission / absorption of light through a highly blocking optical filter. To prevent light from the excitation source from hitting the detector and thus being stronger than the actual signal to be measured, the combined optical density of all filters located in the detection path must be sufficiently high. The exact amount of the optical density depends on the modality, sample, bleaching etc.
The following numbers serve the user as a rough guideline in choosing the right filter. Optimization and experimental verification are up to the user and the exact experiment which is conducted. Bleaching of the sample usually requires much higher OD numbers!
Note: Widefield microscopy:
Fluorescence efficiency is approximately 10-2 to 10-3 with respect to the excitation light intensity (obviously also depending on the sample and its bleaching status!) -> minimum of OD 3-4 for widefield filters (dichroic + emission filter).
Note: Two-Photon microscopy
CONFIDENTIAL PROSPECTIVE INSTRUMENTS Rev. 24-02-13 Page 9 of 17
Two-Photon fluorescence efficiency is very low, can be 10-5 to 10-6 with respect to the femtosecond laser excitation intensity. -> minimum of OD 6 (dichroic + laser blocking + emission filters).
Note: Three-Photon microscopy
Three-Photon fluorescence efficiency is very low, can be 10-7 to 10-8 with respect to the femtosecond laser excitation intensity. -> minimum of OD 8 (dichroic + laser blocking + emission filters).
Note: Forward-direction and epi-direction detection:
Since the fluorescence efficiency in epi (backward) direction is much lower than in forward direction for SHG and THG imaging, it may be advisable to further increase the optical density of the filters. Especially for THG imaging, epi-detection usually provides a very low fluorescence signal efficiency.
What is the penetration depth of your microscope?
This is heavily dependent on the sample. With cleared or transparent samples, the penetration depth is mainly limited by the working distance of the used objective. Multi- mm deep images can be easily acquired in this case. Highly dense, non-cleared samples with large and dense scattering areas will reduce the penetration depth and might end at a depth of 200 – 300 μm.
Is optogenetics possible?
Yes, ScanImage supports optogenetics and an optogenetics beam path can be added to the MPX or MPC 360-Frontend.
Is 3P imaging also possible?
The MPC 3P-ready microscope body supports 3P imaging.
Widefield Epifluorescence Imaging
The MPX and MPC incorporate a powerful “classical” epifluorescence widefield imaging modality, which can serve as a standard fluorescence microscope for general purpose lab- routine imaging of all sorts of specimens, like cell cultures, well-plates etc. – both in upright, as well as inverted configuration. WSI images can be taken as well.
Illumination sources
The controller of the MPX/MPC incorporates the illumination source for the widefield modality. It is entirely integrated into the housing and can be fully controlled by Prospective Instruments ChromoGazer software. A 3mm liquid light guide guides the illumination into the 360-Frontend.
What objectives can be used in your microscope?
All infinity corrected objectives (Nikon, Olympus, Thorlabs) can be used for the widefield path – others (Zeiss, Leica) will change the FOV. For the multiphoton imaging, the objective should be transmissive in the near infrared (up to 1000 nm for two-photon and up to 1700 nm for three-photon). Moreover, the resolution for non-linear microscopy techniques like two-photon depends on the NA and not the magnification! So please use objectives with a high NA!The thread in the objective turret is M32 x 0.75 for all 3 positions.
Prospective Instruments offers the integration of any customer specific illumination source, but typically provides the following choices for the customer to order:
- White-light LED based high power >3W source.
- 7-channel LED based source (395/25, 438/29, 475/28, 511/16, 555/28, 575/25,637nm/12nm).
- 8-channel LED based source (395, 438, 475, 511, 555, 575, 635, 747nm).
- 9-channel LED based source: Selection of 9 channels out of the following ones: 340,378/52, 438/24, 474/27, 509/22, 554/23, 578/21, blank filter 500 – 600nm, 635/18, 735/28, 775/46
External trigger functions, device monitoring and error reporting are provided at the controller back panel or via software functions.
Filters for widefield epifluorescence
The excitation filters are usually built in the illumination source (except for the white-light source) and do not need to be mounted extra inside the 360-Frontend in the fluorescence filter cube.
Dichroic and emission filters for the proper fluorescence & illumination laser line must be placed inside the 360-Frontend. Prospective Instruments supplies either Penta-band, Quad- band or Tipple-band filters to cover that range. The use of multi-band filters allows rapid multispectral measurements without the need to change the filter set.
Motorized filter wheel
Software controlled motorized filter wheel (which can hold up to 8 individual filter cubes) can be realized in the MPC with the Olympus motorized filter wheel solution, whereas in the MPX only a 2-position filter cube “wheel” can be realized.
What cameras do you use for widefield epifluorescence detection?
We use sCMOS cameras from Excelitas, but any other vendor can be integrated.
What field of view (FOV) do you have for widefield epifluorescence?
For our standard water-immersed objectives 16x and 20x the FOV for the widefield epifluorescence is 800 μm and 650 μm respectively.
What is the resolution of the widefield epifluorescence?
For the widefield epifluorescence we use a sCMOS camera with a resolution of 2048×2048 pixels and a pixel size of 6.5 μm x 6.5 μm. With the 20x Olympus water objective this results in a resolution of 0.325 μm per pixel.
Objectives
All infinity-corrected objectives can be used in the widefield modality.
ADAPTIVE OPTICS (AO)
The MPX and MPC currently support the use of the deformable phase plate adaptive optics module Delta7 from the company Phaseform.
It can easily be installed right at the objective threads without any modification of the MPX or MPC.
Samples & Motion Control
What kind of samples can be imaged with your MPX microscopes?
All biological samples, even living animals.
Which fluorophores fit to 1030-nm imaging?
In general, all red-shifted fluorophores fit, like all derivates of RFPs.
Calibration of the FOV and mosaic scanning
We offer a calibration slide, based on a micromachined LiNbO3 wafer with a defined grid pattern. LiNbO3 shows a strong SHG signal, does not photo bleach and can sustain high peak powers from your laser.
Alternatively, we would recommend the R1L3S3P slide from Thorlabs combined with a fluorescent slide underneath. However, care must be taken not to destroy the slide.
Can the microscope controller be equipped with a second 360- Frontend?
From an engineering perspective, a second 360-Frontend could be attached to the microscope controller, driven by the same laser engine, power supplies etc. as the first 360- Frontend. However, galvo-control and data acquisition cannot be shared between two 360- Frontends, therefore resulting in only rather limited advantage in price and complexity.
Motion control & stages
If no motion control is ordered, we provide a rigid fixed stand for the microscope about 300mm in height.
However, motion control can be highly customized and is always up for discussion. As the 360-Frontend is connected to the microscope with a flexible umbilical of about 2m length, “any” motion control solution can be realized in that range.
Typically, we provide a 3-axis XYZ motion control system with 100mm travel on each axis and <4m resolution. In addition, we can implement one axis each for pitch and yaw (in total 5 axis).
Nanometer precise z-focus movement can be realized with the integration of piezo-driven objective z-stage (with up to 0.5 mm stroke), typically supplied by the company Piezoconcept. However, any other manufacturers of piezo driven objective focusers can be integrated.
Does the MPX come with manual XY sample stages and how about Z-focus?
No. We typically ship with a rigid stand that holds the 360-Frontend fix in place. In this case, the sample needs to move in XYZ (also z-focus).
Upright and inverted imaging
To change the microscope from upright to inverted imaging (or vice versa), one only needs to mount the 360-Frontend on an adapter plate to flip the 360-Frontend 180 degrees. On top of the adapter plate, microscopy tables from various vendors can be mounted.
Data & Software
ChromoGazer
To control the basic microscope features, modality changes, laser and light sources, system performance, monitoring, error handling and reporting, Prospective Instruments own “ChromoGazer” software is used and installed.
Widefield imaging is also included in ChromoGazer.
ScanImage
Multiphoton laser scanning, multiphoton data acquisition and visualization is handled by ScanImage. It ́s the most advanced software for controlling laser scanning microscopes with over 1100 citations from peer-reviewed publications.
We offer either Basic or Premium versions of ScanImage.
More info can be found here. https://www.mbfbioscience.com/products/scanimage
What file-format do you use?
Images are saved as .tiff-files.
What software do you use?
To operate the microscope ScanImage is used for the multiphoton scanning process. This is MATLAB based software. In addition to that, our own software (ChromoGazer) is needed to image in widefield mode, control the lasers, adjust system settings, and perform error handling. The ChromoGazer also controls the widefield modality and tells you about the system and laser status.
In addition, we use ImageJ for image processing.
What software for image analysis can I use?
We recommend ImageJ / Fiji and Napari. However, all other commercially available tools like Arivis, Aivia, Imaris work as well.
Will this system benefit or require 10G networking or would regular gigabit suffice?
Gigabit is sufficient.
Remote access or on-site access to this device required for installation and maintenance?
Installation of the device is easy and straight forward for any technical orientated person. Remote access can be useful for service or explanatory tasks. We use TeamViewer for that.
Are you performing updates remotely or is that our responsibility, if so, do you have a protocol or information about updates/upgrades?
Windows and antivirus should be doing automatic updates.
How do you distribute future updates, do all customers receive updates or is there a licensing or upgrade fee.
Installation files for updates will be sent to the customer. MATLAB and ScanImage will include updates for one year but can be updated at own expense after one year if required. But it is not essential for running the microscope.
With Windows 10 EOL on the horizon, will there be an upgrade path to Windows 11 soon?
We will either send the device already with Win11 or upgrade it later. This depends on the 3rd party software e.g. ScanImage.
All devices on our network require antivirus protection, and updates to critical security issues. Do you provide that on your end, or can we install our own?
Inside the microscope controller is a high-performance PC and you can configure and install any software you want. Only restriction is that Windows 10 (for now) must run on it. When formatting the PC, we can reinstall MATLAB, ScanImage and the ChromoGazer in a remote session.
Electronics & Controller
Uninterruptable power supply (UPS)
A UPS can be supplied or used, but it is not necessary to protect the laser or the microscope per se.
Power requirements:
- The system is auto-switching from110V – 230V, 50-60 Hz
- Power consumption 800W maximum. In standby (which means no scanning),the complete system consumes <300W.
- Only standard power outlets needed. One for the microscope, one for the display and one for the XYZ-stages (optional). Normal power strip can be used.
- No water cooling required.
Controller of MPX & MPC
The MPX and MPC microscope share the same controller housing.
Imaging Service
How does it work if I want to ship samples to your side?
One can either send it by post or pass by personally.
Can I do the imaging by myself?
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Yes, after some instructions.
Training
User training can be administered and taught very efficiently, as the complete functionality of the microscope is tightly integrated by our own ChromoGazer software. Access to the microscope can be granted by any remote desktop software and users can be trained remotely via Prospective Instruments support engineers.
General
Service & maintenance
Prospective Instruments designs, manufactures and services its instruments. As we also build the femtosecond laser by ourselves, we also control the most critical component in the system. Therefore, there is only one number to call in case of maintenance or malfunction of a component and a quick response is ensured.
All components of the microscope can be serviced and replaced in the field (including femtosecond laser), thus minimizing downtime.
MPX Microscopes
How does the MPX-series work?
The tight integration of the required light engines (femtosecond laser, 8-channel cw light engine, white light source) together with the corresponding detection modules and beam handling optics allows an entirely new way of implementing and using a multi modal microscope. The microscope becomes a truly turn-key, integrated, compact, easy-to-use, maintenance-free and very easy-to-install imaging platform.
The user does not need to worry about laser alignment, detection efficiency or non-compatible or misaligned hardware over time. The entire microscope consists of two parts, controlled through a flexible, non-detachable umbilical, the scanhead and the controller box. The compact scanhead incorporates laser beam handling and scanning, detectors and control electronics. Everything is pre-aligned, rigid and firmly fixed. The scanhead can be rotated and translated in any direction (truly 3D) and enables all multi-photon-based imaging needs. Cells, molecules, even live whole animal imaging can be accomplished.
The controller on wheels incorporates a built-in PC together with other electronic and photonic devices. Only a power cord, keyboard, mouse and display need to be connected to the controller box to make it completely operational. The device can easily be operated even by non-experts. It allows, through the built-in multimodality, extra features and combination of imaging capabilities for correlative microscopy which is not available until now. It is simple to use, and has the utility of a household appliance. Researchers can share the microscope; it is easily moveable and transportable between different locations. This novel capability is unique to the field, and serving a growing need. A modular and flexible product layout allows the user to upgrade the microscope at a later stage and ensures the researcher can add extra features over time on the same platform.
What multi-photon modalities are integrated in your imaging platform?
Currently, you have the possibility to image in widefield fluorescence, two-photon fluorescence and brightfield modality without changing the device.
What is the imaging workflow of the product?
The user can freely choose between the built-in modalities (1P, 2P and brightfield) to image their samples with a broad spectrum of corresponding modalities. The options are limitless. They can then easily overlay/correlate images and process the multidimensional/multimodal data with our powerful software.
Who can use your product?
- University labs in the biomedical and neuroscience sector
- Hospitals with a strong R&D background
- The cosmetics, pharmaceutical, diagnostics and clinical CRO (contract research organization) industry sector
- Forensic R&D could also be a potential target audience.
- MDs in the hospitals, working with patients deploying intra operating theatre imaging replacing or enhancing classical HE staining and immunohistochemistry (#instantpathology).
What are your target imaging applications?
- Label-free imaging
- Digital pathology
- Whole slide imaging (WSI)
- Deep tissue imaging
- 3D imaging
- Imaging of fresh biopsies
- Immunohistochemistry (IHC), Immunocytochemistry (ICC) and other labelling techniques
- Life cell imaging
- Organoid imaging
- Neuroimaging
- Live animal imaging
- Whole animal imaging
- Calcium monitoring
Is the wavelength of the laser mainly designed for SHG/THG? Most customers who are using GFP and GCamp expect the wavelength to be 900-950nm. Is the wavelength of built-in laser selectable?
The laser wavelength is fixed at around 1040nm for now. SHG and 3PEF works, for THG the laser wavelength is still a little bit too short, as THG will be generated too far in the UV range (although it will work in forward direction).
Are there emitting filters for multi-color in two-photon-mode required?
Yes, one can use up to 4 PMT channels simultaneously with the appropriate bandpass and dichroic filters.
The power of the FSX laser is >400 mW. Is it enough for imaging?
Typical loss in the scan optics and objective account for approx. 50 % which leaves about 200 mW at the sample.
Does the galvo scan software support other resolution modes, such as 256x256?
Yes, we are using ScanImage as scan software. The lowest resolution mode is 64×64.
Is there a resonant scanner option?
Yes, there is the possibility to add a resonant scanner.
Does 2P-mode support objectives with higher NA?
Yes, we have tested Nikon 16x/0.8 NA and Olympus 25x/1.0 NA. The higher the NA, the better!
As for 16x and 4x objectives, are they both available in 2P and fluorescence, or only in separate mode?
Our software allows to choose independently any of the 3 positions on the turret. The Customer can mount any objective on any position.
Does the motorized manipulator support tilting the microscope body and moving the objective for imaging of non-horizontal surfaces and volumes such as temporal lobe observation?
As the scanhead is freely movable (translation & rotation) in all directions, one can move along any axis.
During shipping, will the light path change and require another calibration?
There will be no re-alignment necessary. Our microscope will arrive turn-key at your facility.
Do 2P-fluorescence and wide field belong to the standard configuration, or are they optional?
These modalities are optional.
Can this microscope do optogenetics stimulation?
No, not yet.
Can the customer upgrade the configuration of the microscope or computer?
Yes, the configuration can be updated anytime.
Can we overlap images?
Yes, we can provide you with the according solution.
Is there a time limitation for continuous recording?
No, there is no limitation. The system is properly air cooled. In fact, our demo device is often under test for a long time without problems.
What is the highest working space?
There is basically no limit to the working space. The umbilical connecting scanhead and controller is 2 meters long.
How long does it take to image a 1x1 cm sample?
Typically 10 minutes. However, the scan speed depends on dwell time, resolution and objective.
Does the surface of the sample need to be flat for image acquisition?
Ideally it should be flat. If we get 3-dimensional tissues samples, we gently press them against the glass bottom, of the Fluorodish (or any other) plate.
Do you offer service? How can we get a service technician?
Yes, we do. Please contact us for further information.
What is the field of view for 2P-microscopy?
Depends on the galvo-scanangle and objective: You can choose between 60 µm and up to 1200 µm
Can I use conventional fluorescence dyes for 2P-microscopy?
Yes, you can use standard fluorescence dyes and use filter-sets optimized for your spectral range. In addition, you can optimize your experiments by using special commercially available 2P high-absorbance fluorescence dyes.
What is the file-format of the images?
You can save the images as tiff-files and pre-processing the RGB-images (Brightfield) or 16-bit grayscale (1PM and 2PM) images with the image editing software of your choice.
Do I need programming-skills to use your microscopes?
No, we are working with well-established software like Windows10, ScanImage, µManager, Fiji or MATLAB. All software will be pre-installed and ready-to-use.
Does the resonant galvo-galvo option also allow scanning at the slower rate offered by the galvo-galvo scanner?
Yes, this is no problem. The customer can choose between galvo-galvo only or with resonant.
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