After rigorous preparation, your cells will need a micro-observation environment that is conducive to their viability, compatible with your experiments protocol and all techniques of microscopy. The Bioptechs Chamber Systems were designed with these specifications in mind.

Bioptechs offers three Environmental Chamber models

• The FCS2 Closed System for Inverted Microscopes

• The FCS3 Closed System for Upright Microscopes

• The Delta T Open Dish System for Upright or Inverted Microscopes

Closed System Micro-Observation

Closed systems are used when the experiment requires that the specimen be contained in a completely air tight environment or the optical constraints of the microscope dictate a more precise definition of optical surfaces.

The Focht Chamber System is a closed, live-cell micro-observation chamber that offers several advantages over other chambers. In addition to its unique perfusion and thermal control systems, it provides compatibility with all modes of microscopy, uniform temperature control throughout the observation aperture, a near laminar flow that is adjustable to provide the user with the ability to modify the separation of optical surfaces, flow path geometry, and fluid volume.

Attributes

• Suitable for no flow through high rate flow procedures where a rapid exchange of media is required with low cell surface shear
• Cell temperature can be controlled from ambient to 50 degrees C (±0.2 degrees C) without the need of an air curtain
• Temperature is controlled uniformly across entire field with media equilibrating as it enters the chamber
• Closed system so that bicarbonate CO₂ or organic buffers can be employed
• Compatible with 1/16" tubing for perfusion (C-Flex, Tygon, etc.)
• Easily assembled with ordinary skill (no tools required)
• Stand-alone temperature controller with an alarm circuit to safeguard your cells
• Near laminar flow

The systems are typically comprised of:

• Chamber (environmental optical cavity)
• Electronic Controller
• Stage Adapter
• Objective Heater
• Objective Temperature Controller

FCS2 Chamber	FCS3 Chamber	Objective Heater

How does it work?

Temperature Control

The Focht Chamber Systems were designed to maintain accurate thermal control and allow high volume near laminar flow perfusion. Both of these functions are incorporated into Bioptechs' patented Microaqueduct Slide (see drawing below). The surface of the slide, opposite the specimen side, is coated with an electrically conductive transparent thin film of Indium-Tin Oxide (ITO) and two electrical contacts (bus bars). When the FCS is completely assembled and placed on the stage adapter, two electrical contacts and a thermal sensor contained in the electrical enclosure (not shown in drawing), rest on the bus bars. A temperature controller is used to pass a regulated current flow through the ITO Coating. This causes the surface of the slide to heat. The heat is transferred through the perfusable media to the cell surface on the coverslip thereby providing a conductive heat transfer. The metal base of the chamber is also temperature regulated to provide heat to both the incoming media and peripheral thermal support to the metal housing.

Perfusion

A fluid pathway is formed by separating the Microaqueduct slide from the coverslip containing cells with a single silicone gasket. This gasket can be any thickness from 50 micron to 1mm and any lateral geometry you choose or create. This arrangement allows the user to define the flow characteristics. Therefore, you are not limited by the geometry of the optical cavity instead you select or create it! Fluid access to this flow channel is made through two 14-gauge needle stock tubes protruding from the sides of the chamber top. These tubes provide fluid connection to two perfusion holes in the Microaqueduct slide that interface two "T" shaped grooves cut into the inner surface of the Microaqueduct slide. The “T” groove allows the media to seek the path of least resistance and become nearly laminar before flowing across the cells. This technique eliminates the need for the metal perfusion ring and additional gaskets, which are the limiting factors, required by most conventional chambers.

Microaqueduct design enables proper Koehler illumination with high numeric aperture optics for both transmitted and reflected modes of microscopy:

Top View of Optical Cavity

The drawing, below, is a representation of the Microaqueduct Slide of the FCS3 system. Isometric View of Optical Cavity Microaqueduct Slide is transparent glass but rendered as a solid to improve contrast to illustrate function.

Specifications

FCS2 Exploded View

Electrical Enclosure (can also be detached to sterilize the perfusion tubes) Temperature sensor Heater contacts Upper Half Contains the perfusion tubes Perfusion Tubes (14 gauge) Upper Gasket Microaqueduct Slide (an optical surface which integrates perfusion and temperature control) High volume laminar flow Koehler Illumination Electronically conductive coating for temperature control Singular Lower Gasket This gasket can have any internal geometry you desire Standard thicknesses from 0.1 mm to 1 mm Allows you to define the volume and flow characteristics of the chamber 40 mm Coverslip Self-locking Base (designed to assure parallel uniform closure, eliminate leaks, and broken coverslips) Temperature controlled Dovetail mounted to scope for stability No tools for assembly

FCS3 Exploded View

FCS3 Chamber Top FCS3 Pressure Plate 40mm Coverslip Upper Gasket Thickness and internal geometry allows user to define flow characteristics Standard thicknesses from 0.1 mm to 1 mm Microaqueduct Slide (An optical surface that integrates perfusion and temperature control) Near laminar flow Koehler Illumination compatible Electronically conductive coating for temperature control Lower Gasket Seals chamber base to Microaqueduct slide Perfusion Tubes (14 gauge) Compatible with 1/16th inch tubing FCS3 Chamber Base (Designed to assure parallel uniform closure, eliminate leaks, and broken coverslips) Temperature controlled No tools for assembly

By simply changing this one gasket you can change the volume of the chamber. This gasket can have any internal geometry you desire and can be any thickness from 0.1 mm to 1 mm. The drawing below shows the standard shapes of the gaskets that we include with every FCS. Also included are solid gaskets for you to custom fit to your application. Once you have found the shape that works best for your experiment, you can contact Bioptechs to have a die made to those specifications. Examples of standard gasket outlines (below):

Custom shapes are available. Contact Bioptechs to make arrangements for their production.

It does not matter what chamber you use, if you are using immersion objectives on mammalian specimens, you will need an

The Problem

When live-cell imaging requires the use of high numeric aperture lenses, it is necessary to control the temperature of the objective as well. This problem exist because the optical coupling medium (oil, glycerin, or water) acts as a thermal coupling medium and draws heat away from the specimen. The thermal mass of a fluid coupled objective is overwhelming when compared to the thermal mass of the cells. Unfortunately microscope manufacturers do not offer temperature controlled objectives, nor do they consider the need for thermal regulation in the design of their objectives. Bioptechs has developed and patented a product that solves this problem for the majority of immersion objectives.

The Solution

To eliminate this thermal gradient, Bioptechs offers a patented Objective Heater System which includes a heater/sensor and an electronic controller. The heater/sensor is comprised of an adjustable thin film heating band which surrounds 3/4 of the diameter of the upper region of the central retracting tube of the objective. A surface probe thermal sensor positioned in the gap formed between the ends of the heating band measures the temperature of the objective. This heater/sensor assembly is supported on an adjustable metal mounting to fit objectives ranging from 17 to 35 mm in diameter. The heater loop requires a minimum of 3.5 to 6 mm longitudinal, physical contact with the cylindrical objective surface. In some cases there may be a decorative collar on the objective which must be removed in order to permit adequate surface contact.

Specifications

The Controller is specifically designed to slowly heat the objective over a fifteen minute warmup period then hold the objective at the setpoint value within 0.2 degrees C. The Controller operates from ambient to 50 degrees C and has special safety circuitry which utilizes a 0.9 degrees C error window to shut down the controller and sound an alarm if, for any reason, the temperature of the objective deviates after it has reached setpoint. A user calibration test is also built-in to the controller.

Special note

The Bioptechs Objective Heater fits most, but not all, high N.A. objectives. Due to the size and geometry of some objectives, a direct contact heater cannot be used.

To determine if a contact heater can be used, confirm that there is a longitudinal 5 mm high cylindrical exposed region on the central retracting tube near the specimen end of the objective. If there is access to this geometry, and its diameter is between 17 to 35 mm a Bioptechs objective heater can be used.

Although there have been no studies relative to the effects of thermal cycling of objectives between ambient and 37 degrees C, Bioptechs recommends storing of warmed objectives in a constant temperature enclosure when not in use. The Boekel desktop incubator is well suited for this application as well as a convenient place for temporary storage of specimens or media near your microscope.

When using the Bioptechs Objective Heater, Cargille type 37 Immersion oil is highly recommended to optimize the objective's PSF. This oil is formulated to have a refractive index of 1.515 at 37 degree C. This is an ideal optical coupling medium for in-vitro, live-cell imaging (not for in-vivo use).

Closed-System Cooled Chamber

How it works

Cells are plated on a 40mm coverslip and placed into a special FCS2 chamber. This provides a perfuseable laminar flow optical chamber with user modifiable flow characteristics. The upper glass element (microaqueduct slide) is then used to remove heat from the specimen cavity to be absorbed in the cooled fluid being circulated in the cavity formed by the addition of an O-ring sealed window adapter. Gravity or an electric pump is then used to create a flow of chilled fluid through the heat exchange cavity in the chamber. The cells remain safely enclosed in a separate optical enclosure. If more thermal transfer is necessary, tubing can be installed into the FCS2 base.

Note: If you are using high N.A. objectives at below ambient temperatures it will be necessary to cool and thermally isolate the objective from the scope. The optional thermal isolator will prevent condensation from forming on the lower element of the objective. A gas port is provided to purge the adapter with dry nitrogen. An anti-reflection coated window optimized for 340-700nm seals the bottom of the adapter.

Objective Cooling Collar

The Objective Cooling Collar is an isolated fluid and thermal transfer device that attaches to the top of the objective and provides a means of attaching coolant lines from a cold sourse or chiller bath. The removal of energy from the objective is more difficult and less efficient than heating. Make sure you have enough substage room for the additional diameter required for the cooling collar and tubing. Cooling collars are precision machined to fit specific objectives. Please contact Bioptechs for additional compatibility information.

An open dish can be used when you need to have physical access to cells or tissue for such applications as microinjection, electrostimulation, wounding, micromanipulation, use of mechanical probes, recording, and stimulation. -or- An open system may offer the most economical, convenient, and accurate micro-observation method for your application providing an open-fluid surface is acceptable.

Delta T Temperature-Controlled Culture Dish System

The Bioptechs Delta T is a culture dish system specifically designed for live-cell microscopy! Now you can have accurate temperature control and high-numeric aperture compatibility in a convenient disposable culture dish system that even works in confocal applications.

• Easily adapted to a variety of specimen types from monolayered adherent cells to brain slice and tissue preps
• Low mass to thermo-regulate as opposed to conventional stage heaters
• Plate, incubate, and observe without the need to transfer your cells
• Fast thermal recovery after perfusion (within seconds)
• Compatible with inverted and upright microscope stands
• Coverglass bottom for optimum optical compatibility
• No need for warm air blowers or stage heaters
• Direct first-surface heating to your cells
• Can also be cooled below ambient
• Perfusion available (Perfusion Configuration)
• No preheating

The Delta T Culture Dish System is designed to simulate host conditions on the stage of your microscope and provide an optimal optical environment for microscopy. This two-step system allows you to plate your cells and observe them without having to transfer them to another structure. The system components are, the Controller, Stage Adapter, and Dishes. Accessories for Tissue Slice, Brain Slice, and other specimens are available.

An intelligent feedback loop passes an electrical current through a thin film coating on the underside surface of the glass substrate on which the cells are grown. Heat is applied directly to the cells without the inefficiencies associated with peripheral heating by traditional culture dish warmers. Bioptechs exclusively offers opaque culture dishes which eliminates the unwanted ambient light background for fluorescence imaging.

The controller features a real-time temperature display and fast learning curve to compensate for cooling due to surface evaporation while responding to temperature changes due to perfusion. There is also an alarmed protection circuit to safeguard the cells and an internal reference for the user adjustable calibration. The standard controller has a temperature range of ambient to 50 degrees C. Extended ranges are available upon request.

Delta T dishes have 35 mm O.D. and a 23 mm central aperture. The peripheral region of the dish is tapered to reduce the dead-volume and the height of the dish is 6 mm to allow better access for micro-injection and micromanipulation. The dishes are a hybrid of polystyrene plastic and Desag 263 glass. The outer structure of the dish is available in opaque black or clear, and come with a clear 0.5 mm or #1.5 glass coverslip bottom bonded to it. The dishes are also available in a plain-glass unheated version.

Low numeric aperture users need only purchase the basic Delta T with 0.5 mm dishes. However if you are using high numeric aperture objectives (immersion) you will need the 0.15 mm dishes. It will be necessary to regulate the temperature of the objective as well. An Objective Heater and an Objective Heater Controller will be necessary for uniform temperature across the field.